Regulation of human phosphatidylinositol-specific phospholipase c-like enzyme

ABSTRACT

Reagents which regulate human phosphatidylinositol-specific phospholipase C-like enzyme and reagents which bind to human phosphatidylinositol-specific phospholipase C-like enzyme gene products can play a role in preventing, ameliorating, or correcting dysfunctions or diseases including, but not limited to, cancer, peripheral and central nervous system diseases, and chronic obstructive pulmonary disease.

TECHNICAL FIELD OF THE INVENTION

[0001] The invention relates to the area of enzyme regulation. More particularly, the invention relates to human phosphatidylinositol-specific phospholipase C-like enzyme and its regulation.

BACKGROUND OF THE INVENTION

[0002] Phospholipase C (PLC) is an enzyme which hydrolyzes glycerophospholipid and sphingophospholipid. See U.S. Pat. No. 6,060,302. PLC is present in the spleen, the tunica mucosa interstini tenuis, and the placenta, of mammals and plays an important role in mammalian metabolism. For example, phosphatidylinositol-specific phospholipase C (PI-PLC) hydrolyzes phosphatidylinositol 4,5-diphosphate to generate 1,2-diacylglycerol and inositol 1,4,5-triphosphate (Rhee et al., Science 244, 546-50, 1989). Various PLC isozymes have been described (e.g., Bennett et al., Nature 334, 268-70, 1988; Emori et al., J. Biol. Chem. 264, 21885-90, 1989; Katan et al., Cell 54, 171-77, 1988); Ohta et al., FEBS Lett. 242, 31-35, 1988; Stahl et al., Nature 332, 269-72, 1988; Suh et al., Cell 54, 161-69, 1988; and Kritz et al., CIBA Found. Symp. 150, 112-27, 1990). Because of the importance of PLC in mammalian metabolism, there is a need in the art to identify additional PLC-like enzymes which can be regulated to provide therapeutic effects.

SUMMARY OF THE INVENTION

[0003] It is an object of the invention to provide reagents and methods of regulating a human phosphatidylinositol-specific phospholipase C-like enzyme. This and other objects of the invention are provided by one or more of the embodiments described below.

[0004] One embodiment of the invention is a PI-PLC-like enzyme polypeptide comprising an amino acid sequence selected from the group consisting of:

[0005] amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 2; and

[0006] the amino acid sequence shown in SEQ ID NO: 2.

[0007] Yet another embodiment of the invention is a method of screening for agents which decrease extra-cellular matrix degradation. A test compound is contacted with a PI-PLC-like enzyme polypeptide comprising an amino acid sequence selected from the group consisting of:

[0008] amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 2; and

[0009] the amino acid sequence shown in SEQ ID NO: 2.

[0010] Binding between the test compound and the PI-PLC-like enzyme polypeptide is detected. A test compound which binds to the PI-PLC-like enzyme polypeptide is thereby identified as a potential agent for decreasing extra-cellular matrix degradation. The agent can work by decreasing the activity of the PI-PLC-like enzyme.

[0011] Another embodiment of the invention is a method of screening for agents which decrease extra-cellular matrix degradation. A test compound is contacted with a polynucleotide encoding a PI-PLC-like enzyme polypeptide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of:

[0012] nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1;

[0013] the nucleotide sequence shown in SEQ ID NO: 1;

[0014] nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 4; and

[0015] the nucleotide sequence shown in SEQ ID NO: 4.

[0016] Binding of the test compound to the polynucleotide is detected. A test compound which binds to the polynucleotide is identified as a potential agent for decreasing extra-cellular matrix degradation. The agent can work by decreasing the amount of the PI-PLC-like enzyme through interacting with the PI-PLC-like enzyme mRNA.

[0017] Another embodiment of the invention is a method of screening for agents which regulate extra-cellular matrix degradation. A test compound is contacted with a PI-PLC-like enzyme polypeptide comprising an amino acid sequence selected from the group consisting of:

[0018] amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 2; and

[0019] the amino acid sequence shown in SEQ ID NO: 2.

[0020] A PI-PLC-like enzyme activity of the polypeptide is detected. A test compound which increases PI-PLC-like enzyme activity of the polypeptide relative to PI-PLC-like enzyme activity in the absence of the test compound is thereby identified as a potential agent for increasing extra-cellular matrix degradation. A test compound which decreases PI-PLC-like enzyme activity of the polypeptide relative to PI-PLC-like enzyme activity in the absence of the test compound is thereby identified as a potential agent for decreasing extra-cellular matrix degradation.

[0021] Even another embodiment of the invention is a method of screening for agents which decrease extra-cellular matrix degradation. A test compound is contacted with a PI-PLC-like enzyme product of a polynucleotide which comprises a nucleotide sequence selected from the group consisting of:

[0022] nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1;

[0023] the nucleotide sequence shown in SEQ ID NO: 1;

[0024] nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 4; and

[0025] the nucleotide sequence shown in SEQ ID NO: 4.

[0026] Binding of the test compound to the PI-PLC-like enzyme product is detected. A test compound which binds to the PI-PLC-like enzyme product is thereby identified as a potential agent for decreasing extra-cellular matrix degradation.

[0027] Still another embodiment of the invention is a method of reducing extra-cellular matrix degradation. A cell is contacted with a reagent which specifically binds to a polynucleotide encoding a PI-PLC-like enzyme polypeptide or the product encoded by the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of:

[0028] nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1;

[0029] the nucleotide sequence shown in SEQ ID NO: 1;

[0030] nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 4; and

[0031] the nucleotide sequence shown in SEQ ID NO: 4.

[0032] PI-PLC-like enzyme activity in the cell is thereby decreased.

[0033] The invention thus provides a human PI-PLC-like enzyme which can be used to identify test compounds which may act, for example, as activators or inhibitors at the enzyme's active site. Human PI-PLC-like enzyme and fragments thereof also are useful in raising specific antibodies which can block the enzyme and effectively reduce its activity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034]FIG. 1 shows the DNA-sequence encoding a PI-PLC-like enzyme polypeptide (SEQ ID NO:1).

[0035]FIG. 2 shows the amino acid sequence deduced from the DNA-sequence of FIG. 1 (SEQ ID NO:2).

[0036]FIG. 3 shows the amino acid sequence of the protein identified with SwissProt Accession No. P34024 (SEQ ID NO:3).

[0037]FIG. 4 shows the DNA-sequence encoding a PI-PLC-like enzyme polypeptide (SEQ ID NO:4).

[0038]FIG. 5 shows the BLASTP alignment of PI-PLC-like enzyme polypeptide (SEQ ID NO:2) with the protein identified with SwissProt Accession No. P34024 (SEQ ID NO:3). Active site residues are indicated in bold.

[0039]FIG. 6 shows the relative expression of PI-PLC-like enzyme polypeptide in respiratory cells and tissues.

[0040]FIG. 7 shows the relative expression of human PI-PLC-like enzyme in various human tissues and the neutrophil-like cell line HL60.

[0041]FIG. 8 shows the expression profiling of PI-PLC-like enzyme in whole-body.

[0042]FIG. 9 shows the expression profiling of PI-PLC-like enzyme in blood and lung.

DETAILED DESCRIPTION OF THE INVENTION

[0043] The invention relates to an isolated polynucleotide encoding a PI-PLC-like enzyme polypeptide and being selected from the group consisting of:

[0044] a) a polynucleotide encoding a PI-PLC-like enzyme polypeptide comprising an amino acid sequence selected from the group consisting of:

[0045] amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 2; and

[0046] the amino acid sequence shown in SEQ ID NO: 2.

[0047] b) a polynucleotide comprising the sequence of SEQ ID NOS: 1 or 4;

[0048] c) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b);

[0049] d) a polynucleotide the sequence of which deviates from the polynucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code; and

[0050] e) a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d).

[0051] Furthermore, it has been discovered by the present applicant that a novel phosphatidylinositol-specific phospholipase C-like enzyme (PI-PLC-like enzyme), particularly a human PI-PLC-like enzyme, is a discovery of the present invention. Human PI-PLC-like enzyme comprises the amino acid sequence shown in SEQ ID NO:2. Human PI-PLC-like enzyme was identified by searching human protein sequences with the protein having the sequence shown in SEQ ID NO:3 and identified with SwissProt Accession No. P34024. A coding sequence for human PI-PLC-like enzyme is shown in SEQ ID NO: 1.

[0052] Human PI-PLC-like enzyme is 27% identical over 209 amino acids to the Listeria monocytogenes protein identified with SwissProt Accession No. P34024 and annotated as “phosphatidylinositol-specific phospholipase C” (FIG. 1). Listeria monocytogenes PI-PLC is an important factor in pathogenesis; its activity is present only in virulent listeria species, where it may participate in lysis of the phagolysosomal membrane. Mol. Microbiol. 5, 367-72, 1991.

[0053] Human PI-PLC-like enzyme of the invention is expected to be useful for the same purposes as previously identified phospholipase C enzymes. Thus, human PI-PLC-like enzyme can be used in therapeutic methods to treat disorders such as cancer and COPD. Human PI-PLC-like enzyme also can be used to screen for human PI-PLC-like enzyme activators and inhibitors.

[0054] Furthermore, it has been discovered by the present applicant, that PI-PLC-like enzyme is highly expressed in immune tissues such as spleen, thymus, and peripheral blood leukocytes, and in specific immune cell types such as activated CD4⁺ T cells indicating that PI-PLC-like enzyme plays an important role in inflammation. Similarly, the high expression of PI-PLC-like enzyme in central nervous system tissues such as cerebellum and adult and fetal brain, indicate that PI-PLC-like enzyme plays an important role in cell signaling by neurotransmitters. Since the pathology of asthma is tightly linked to the combined effects of inflammatory mediators that result in inflammation, edema, mucus production, which together chronically thicken the airway walls of the lung, and neurotransmitters that acutely constrict the airways through airway smooth muscle cell stimulation, the modulation of PI-PLC-like enzyme's effects are expected to have beneficial effects in the treatment of asthma.

[0055] Polypeptides

[0056] Human PI-PLC-like enzyme polypeptides according to the invention comprise at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, or 320 contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO:2 or a biologically active variant thereof, as defined below. A PI-PLC-like enzyme polypeptide of the invention therefore can be a portion of a PI-PLC-like enzyme protein, a fill-length PI-PLC-like enzyme protein, or a fusion protein comprising all or a portion of a PI-PLC-like enzyme protein.

[0057] Biologically Active Variants

[0058] Human PI-PLC-like enzyme polypeptide variants which are biologically active, i.e., retain a phospholipase C activity, also are PI-PLC-like enzyme polypeptides. Preferably, naturally or non-naturally occurring PI-PLC-like enzyme polypeptide variants have amino acid sequences which are at least about 50, 55, 60, 65, or 70, preferably about 75, 80, 85, 90, 96, 96, or 98% identical to the amino acid sequence shown in SEQ ID NO:2 or a fragment thereof. Percent identity between a putative PI-PLC-like enzyme polypeptide variant and an amino acid sequence of SEQ ID NO:2 is determined using the Blast2 alignment program (Blosum62, Expect 10, standard genetic codes).

[0059] Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions. Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.

[0060] Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of a PI-PLC-like enzyme polypeptide can be found using computer programs well known in the art, such as DNASTAR software. Whether an amino acid change results in a biologically active PI-PLC-like enzyme polypeptide can readily be determined by assaying for phospholipase C activity, as described for example, in the specific Examples, below.

[0061] Fusion Proteins

[0062] Fusion proteins are useful for generating antibodies against PI-PLC-like enzyme polypeptide amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins which interact with portions of a PI-PLC-like enzyme polypeptide. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.

[0063] A PI-PLC-like enzyme polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond. The first polypeptide segment comprises at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, or 320 contiguous amino acids of SEQ ID NO:2 or of a biologically active variant, such as those described above. The first polypeptide segment also can comprise full-length PI-PLC-like enzyme protein.

[0064] The second polypeptide segment can be a full-length protein or a protein fragment. Proteins commonly used in fusion protein construction include β-galactosidase, β-glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). Additionally, epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP 16 protein fusions. A fusion protein also can be engineered to contain a cleavage site located between the PI-PLC-like enzyme polypeptide-encoding sequence and the heterologous protein sequence, so that the PI-PLC-like enzyme polypeptide can be cleaved and purified away from the heterologous moiety.

[0065] A fusion protein can be synthesized chemically, as is known in the art. Preferably, a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology. Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from SEQ ID NO:1 in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art. Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, Wis.), Stratagene (La Jolla, Calif.), CLONTECH (Mountain View, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.), MBL International Corporation (MIC; Watertown, Mass.), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).

[0066] Identification of Species Homologs

[0067] Species homologs of human PI-PLC-like enzyme polypeptide can be obtained using PI-PLC-like enzyme polypeptide polynucleotides (described below) to make suitable probes or primers for screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which encode homologs of PI-PLC-like enzyme polypeptide, and expressing the cDNAs as is known in the art.

[0068] Polynucleotides

[0069] A PI-PLC-like enzyme polynucleotide can be single- or double-stranded and comprises a coding sequence or the complement of a coding sequence for a PI-PLC-like enzyme polypeptide. A partial coding sequence for human PI-PLC-like enzyme is shown in SEQ ID NO: 1.

[0070] Degenerate nucleotide sequences encoding human PI-PLC-like enzyme polypeptides, as well as homologous nucleotide sequences which are at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, or 98% identical to the nucleotide sequence shown in SEQ ID NO:1 or its complement also are PI-PLC-like enzyme polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of −12 and a gap extension penalty of −2. Complementary DNA (cDNA) molecules, species homologs, and variants of PI-PLC-like enzyme polynucleotides which encode biologically active PI-PLC-like enzyme polypeptides also are PI-PLC-like enzyme polynucleotides.

[0071] Identification of Polynucleotide Variants and Homologs

[0072] Variants and homologs of the PI-PLC-like enzyme polynucleotides described above also are PI-PLC-like enzyme polynucleotides. Typically, homologous PI-PLC-like enzyme polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known PI-PLC-like enzyme polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions—2×SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2×SSC, 0.1% SDS, 50° C. once, 30 minutes; then 2×SSC, room temperature twice, 10 minutes each homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches. Species homologs of the PI-PLC-like enzyme polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast. Human variants of PI-PLC-like enzyme polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well known that the T_(m) of a double-stranded DNA decreases by 1-1.5° C. with every 1% decrease in homology (Bonner et al., J. Mol. Biol. 81, 123 (1973). Variants of human PI-PLC-like enzyme polynucleotides or PI-PLC-like enzyme polynucleotides of other species can therefore be identified by hybridizing a putative homologous PI-PLC-like enzyme polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO:1 or the complement thereof to form a test hybrid. The melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.

[0073] Nucleotide sequences which hybridize to PI-PLC-like enzyme polynucleotides or their complements following stringent hybridization and/or wash conditions also are PI-PLC-like enzyme polynucleotides. Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.

[0074] Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20° C. below the calculated T_(m) of the hybrid under study. The T_(m) of a hybrid between a PI-PLC-like enzyme polynucleotide having a nucleotide sequence shown in SEQ ID NO:1 or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):

T _(m)=81.5° C.−16.6(log₁₀[Na⁺])+0.41(%G+C)−0.63(%formamide)−600/l),

[0075] where l=the length of the hybrid in basepairs.

[0076] Stringent wash conditions include, for example, 4×SSC at 65° C., or 50% formamide, 4×SSC at 42° C., or 0.5×SSC, 0.1% SDS at 65° C. Highly stringent wash conditions include, for example, 0.2×SSC at 65° C.

[0077] Preparation of Polynucleotides

[0078] A PI-PLC-like enzyme polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids. Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated PI-PLC-like enzyme polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments which comprises PI-PLC-like nucleotide sequences. Isolated polynucleotides are in preparations which are free or at least 70, 80, or 90% free of other molecules.

[0079] Human PI-PLC-like enzyme cDNA molecules can be made with standard molecular biology techniques, using PI-PLC-like enzyme mRNA as a template. Human PI-PLC-like enzyme cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template.

[0080] Alternatively, synthetic chemistry techniques can be used to synthesizes PI-PLC-like enzyme polynucleotides. The degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode a PI-PLC-like enzyme polypeptide having, for example, an amino acid sequence shown in SEQ ID NO:2 or a biologically active variant thereof.

[0081] Extending Polynucleotides

[0082] Various PCR-based methods can be used to extend the nucleic acid sequences disclosed herein to detect upstream sequences such as promoters and regulatory elements. For example, restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.

[0083] Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al., Nucleic Acids Res. 16, 8186, 1988). Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Plymouth, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72° C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template. Another method which can be used is capture PCR, which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al., PCR Methods Applic. 1, 111-119, 1991). In this method, multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.

[0084] Another method which can be used to retrieve unknown sequences is that of Parker et al., Nucleic Acids Res. 19, 3055-3060, 1991). Additionally, PCR, nested primers, and PROMOTERFINDER libraries (CLONTECH, Palo Alto, Calif.) can be used to walk genomic DNA (CLONTECH, Palo Alto, Calif.). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.

[0085] When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5′ regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5′ non-transcribed regulatory regions.

[0086] Commercially available capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products. For example, capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled device camera. Output/light intensity can be converted to electrical signal using appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled. Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.

[0087] Obtaining Polypeptides

[0088] Human PI-PLC-like enzyme polypeptides can be obtained, for example, by purification from human cells, by expression of PI-PLC-like enzyme polynucleotides, or by direct chemical synthesis.

[0089] Protein Purification

[0090] Human PI-PLC-like enzyme polypeptides can be purified from any cell which expresses the enzyme, including host cells which have been transfected with PI-PLC-like enzyme expression constructs. A purified PI-PLC-like enzyme polypeptide is separated from other compounds which normally associate with the PI-PLC-like enzyme polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis. A preparation of purified PI-PLC-like enzyme polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis.

[0091] Expression of Polynucleotides

[0092] To express a PI-PLC-like enzyme polynucleotide, the polynucleotide can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding PI-PLC-like enzyme polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1989.

[0093] A variety of expression vector/host systems can be utilized to contain and express sequences encoding a PI-PLC-like enzyme polypeptide. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.

[0094] The control elements or regulatory sequences are those non-translated regions of the vector—enhancers, promoters, 5′ and 3′ untranslated regions—which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPORT1 plasmid (Life Technologies) and the like can be used. The baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding a PI-PLC-like enzyme polypeptide, vectors based on SV40 or EBV can be used with an appropriate selectable marker.

[0095] Bacterial and Yeast Expression Systems

[0096] In bacterial systems, a number of expression vectors can be selected depending upon the use intended for the PI-PLC-like enzyme polypeptide. For example, when a large quantity of a PI-PLC-like enzyme polypeptide is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene). In a BLUESCRIPT vector, a sequence encoding the PI-PLC-like enzyme polypeptide can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of β-galactosidase so that a hybrid protein is produced. pIN vectors (Van Heeke & Schuster, J. Biol. Chem. 264, 5503-5509, 1989) or pGEX vectors (Promega, Madison, Wis.) also can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.

[0097] In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used. For reviews, see Ausubel et al. (1989) and Grant et al., Methods Enzymol. 153, 516-544, 1987.

[0098] Plant and Insect Expression Systems

[0099] If plant expression vectors are used, the expression of sequences encoding PI-PLC-like enzyme polypeptides can be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6, 307-311, 1987). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used (Coruzzi et al., EMBO J. 3, 1671-1680, 1984; Broglie et al., Science 224, 838-843, 1984; Winter et al., Results Probl. Cell Differ. 17, 85-105, 1991). These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (e.g., Hobbs or Murray, in MCGRAW HILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N.Y., pp. 191-196, 1992).

[0100] An insect system also can be used to express a PI-PLC-like enzyme polypeptide. For example, in one such system Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. Sequences encoding PI-PLC-like enzyme polypeptides can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of PI-PLC-like enzyme polypeptides will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which PI-PLC-like enzyme polypeptides can be expressed (Engelhard et al., Proc. Nat. Acad. Sci. 91, 3224-3227, 1994).

[0101] Mammalian Expression Systems

[0102] A number of viral-based expression systems can be used to express PI-PLC-like enzyme polypeptides in mammalian host cells. For example, if an adenovirus is used as an expression vector, sequences encoding PI-PLC-like enzyme polypeptides can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome can be used to obtain a viable virus which is capable of expressing a PI-PLC-like enzyme polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. 81, 3655-3659, 1984). If desired, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.

[0103] Human artificial chromosomes (HACs) also can be used to deliver larger fragments of DNA than can be contained and expressed in a plasmid. HACs of 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).

[0104] Specific initiation signals also can be used to achieve more efficient translation of sequences encoding PI-PLC-like enzyme polypeptides. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding a PI-PLC-like enzyme polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals (including the ATG initiation codon) should be provided. The initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et al., Results Probl. Cell Differ. 20, 125-162, 1994).

[0105] Host Cells

[0106] A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed PI-PLC-like enzyme polypeptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a “prepro” form of the polypeptide also can be used to facilitate correct insertion, folding and/or function. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, Va. 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein.

[0107] Stable expression is preferred for long-term, high-yield production of recombinant proteins. For example, cell lines which stably express PI-PLC-like enzyme polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced PI-PLC-like enzyme sequences. Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. See, for example, ANIMAL CELL CULTURE, R.I. Freshney, ed., 1986.

[0108] Any number of selection systems can be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11, 223-32, 1977) and adenine phosphoribosyltransferase (Lowy et al., Cell 22, 817-23, 1980) genes which can be employed in tk⁻ or aprt⁻ cells, respectively. Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. 77, 3567-70, 1980), npt confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin et al., J. Mol. Biol. 150, 1-14, 1981), and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murray, 1992, supra). Additional selectable genes have been described. For example, trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. 85, 8047-51, 1988). Visible markers such as anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al., Methods Mol. Biol. 55, 121-131, 1995).

[0109] Detecting Expression

[0110] Although the presence of marker gene expression suggests that the PI-PLC-like enzyme polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding a PI-PLC-like enzyme polypeptide is inserted within a marker gene sequence, transformed cells containing sequences which encode a PI-PLC-like enzyme polypeptide can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding a PI-PLC-like enzyme polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the PI-PLC-like enzyme polynucleotide.

[0111] Alternatively, host cells which contain a PI-PLC-like enzyme polynucleotide and which express a PI-PLC-like enzyme polypeptide can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein. For example, the presence of a polynucleotide sequence encoding a PI-PLC-like enzyme polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding a PI-PLC-like enzyme polypeptide. Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding a PI-PLC-like enzyme polypeptide to detect transformants which contain a PI-PLC-like enzyme polynucleotide.

[0112] A variety of protocols for detecting and measuring the expression of a PI-PLC-like enzyme polypeptide, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on a PI-PLC-like enzyme polypeptide can be used, or a competitive binding assay can be employed. These and other assays are described in Hampton et al., SEROLOGICAL METHODS: A LABORATORY MANUAL, APS Press, St. Paul, Minn., 1990) and Maddox et al., J. Exp. Med. 158, 1211-1216, 1983).

[0113] A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding PI-PLC-like enzyme polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, sequences encoding a PI-PLC-like enzyme polypeptide can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

[0114] Expression and Purification of Polypeptides

[0115] Host cells transformed with nucleotide sequences encoding a PI-PLC-like enzyme polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode PI-PLC-like enzyme polypeptides can be designed to contain signal sequences which direct secretion of soluble PI-PLC-like enzyme polypeptides through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound PI-PLC-like enzyme polypeptide.

[0116] As discussed above, other constructions can be used to join a sequence encoding a PI-PLC-like enzyme polypeptide to a nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.). Inclusion of cleavable linker sequences such as those specific for Factor Xa or enterokinase (Invitrogen, San Diego, Calif.) between the purification domain and the PI-PLC-like enzyme polypeptide also can be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing a PI-PLC-like enzyme polypeptide and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by IMAC (immobilized metal ion affinity chromatography, as described in Porath et al., Prot. Exp. Purif. 3, 263-281, 1992), while the enterokinase cleavage site provides a means for purifying the _PI-PLC-like enzyme polypeptide from the fusion protein. Vectors which contain fusion proteins are disclosed in Kroll et al., DNA Cell Biol. 12, 441-453, 1993.

[0117] Chemical Synthesis

[0118] Sequences encoding a PI-PLC-like enzyme polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers et al., Nucl. Acids Res. Symp. Ser. 215-223, 1980; Horn et al. Nucl. Acids Res. Symp. Ser. 225-232, 1980). Alternatively, a PI-PLC-like enzyme polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al., Science 269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of PI-PLC-like enzyme polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.

[0119] The newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., New York, N.Y., 1983). The composition of a synthetic PI-PLC-like enzyme polypeptide can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, supra). Additionally, any portion of the amino acid sequence of the PI-PLC-like enzyme polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.

[0120] Production of Altered Polypeptides

[0121] As will be understood by those of skill in the art, it may be advantageous to produce PI-PLC-like enzyme polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.

[0122] The nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter PI-PLC-like enzyme polypeptide-encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences. For example, site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.

[0123] Antibodies

[0124] Any type of antibody known in the art can be generated to bind specifically to an epitope of a PI-PLC-like enzyme polypeptide. “Antibody” as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab′)₂, and Fv, which are capable of binding an epitope of a PI-PLC-like enzyme polypeptide. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.

[0125] An antibody which specifically binds to an epitope of a PI-PLC-like enzyme polypeptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art. Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody which specifically binds to the immunogen.

[0126] Typically, an antibody which specifically binds to a PI-PLC-like enzyme polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay. Preferably, antibodies which specifically bind to PI-PLC-like polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate a PI-PLC-like enzyme polypeptide from solution.

[0127] Human PI-PLC-like enzyme polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, a PI-PLC-like enzyme polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol). Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially useful.

[0128] Monoclonal antibodies which specifically bind to a PI-PLC-like enzyme polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al., Nature 256, 495-497, 1985; Kozbor et al., J. Immunol. Methods 81, 31-42, 1985; Cote et al., Proc. Natl. Acad. Sci. 80, 2026-2030, 1983; Cole et al., Mol. Cell Biol. 62, 109-120, 1984).

[0129] In addition, techniques developed for the production of “chimeric antibodies,” the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison et al., Proc. Natl. Acad. Sci. 81, 6851-6855, 1984; Neuberger et al., Nature 312, 604-608, 1984; Takeda et al., Nature 314, 452-454, 1985). Monoclonal and other antibodies also can be “humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions. Alternatively, humanized antibodies can be produced using recombinant methods, as described in GB2188638B. Antibodies which specifically bind to a PI-PLC-like enzyme polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. Pat. No. 5,565,332.

[0130] Alternatively, techniques described for the production of single chain antibodies can be adapted using methods known in the art to produce single chain antibodies which specifically bind to PI-PLC-like enzyme polypeptides. Antibodies with related specificity, but of distinct idiotypic composition, can be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton, Proc. Natl. Acad. Sci. 88, 11120-23, 1991).

[0131] Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et al., 1996, Eur. J. Cancer Prev. 5, 507-11). Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol. 15, 159-63. Construction of bivalent, bispecific single-chain antibodies is taught in Mallender & Voss, 1994, J. Biol. Chem. 269, 199-206.

[0132] A nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below. Alternatively, single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar et al., 1995, Int. J. Cancer 61, 497-501; Nicholls et al., 1993, J. Immunol. Meth. 165, 81-91).

[0133] Antibodies which specifically bind to PI-PLC-like enzyme polypeptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al., Proc. Natl. Acad. Sci. 86, 3833-3837, 1989; Winter et al., Nature 349, 293-299, 1991).

[0134] Other types of antibodies can be constructed and used therapeutically in methods of the invention. For example, chimeric antibodies can be constructed as disclosed in WO 93/03151. Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the “diabodies” described in WO 94/13804, also can be prepared.

[0135] Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which a PI-PLC-like enzyme polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.

[0136] Antisense Oligonucleotides

[0137] Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of PI-PLC-like enzyme gene products in the cell.

[0138] Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5′ end of one nucleotide with the 3′ end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol. Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al., Chem. Rev. 90, 543-583, 1990.

[0139] Modifications of PI-PLC-like enzyme gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5′, or regulatory regions of the PI-PLC-like enzyme gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions −10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using “triple helix” base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature (e.g., Gee et al., in Huber & Carr, MOLECULAR AND IMMUNOLOGIC APPROACHES, Futura Publishing Co., Mt. Kisco, N.Y., 1994). An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

[0140] Precise complementarity is not required for successful complex formation between an antisense oligonucleotide and the complementary sequence of a PI-PLC-like enzyme polynucleotide. Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a PI-PLC-like enzyme polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent PI-PLC-like enzyme nucleotides, can provide sufficient targeting specificity for PI-PLC-like enzyme mRNA. Preferably, each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular PI-PLC-like enzyme polynucleotide sequence.

[0141] Antisense oligonucleotides can be modified without affecting their ability to hybridize to a PI-PLC-like enzyme polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule. For example, internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose. Modified bases and/or sugars, such as arabinose instead of ribose, or a 3′, 5′-substituted oligonucleotide in which the 3′ hydroxyl group or the 5′ phosphate group are substituted, also can be employed in a modified antisense oligonucleotide. These modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al., Trends Biotechnol. 10, 152-158, 1992; Uhlmann et al., Chem. Rev. 90, 543-584, 1990; Uhlmann et al., Tetrahedron. Lett. 215, 3539-3542, 1987.

[0142] Ribozymes

[0143] Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al., U.S. Pat. No. 5,641,673). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.

[0144] The coding sequence of a PI-PLC-like enzyme polynucleotide can be used to generate ribozymes which will specifically bind to mRNA transcribed from the PI-PLC-like enzyme polynucleotide. Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. Nature 334, 585-591, 1988). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete “hybridization” region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al., EP 321,201).

[0145] Specific ribozyme cleavage sites within a PI-PLC-like enzyme RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate PI-PLC-like enzyme RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.

[0146] Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease PI-PLC-like enzyme expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. A ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.

[0147] As taught in Haseloff et al., U.S. Pat. No. 5,641,673, ribozymes can be engineered so that ribozyme expression will occur in response to factors which induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.

[0148] Differentially Expressed Genes

[0149] Described herein are methods for the identification of genes whose products interact with human phosphatidylinositol-specific phospholipase C-like enzyme. Such genes may represent genes that are differentially expressed in disorders including, but not limited to, cancer and COPD. Further, such genes may represent genes that are differentially regulated in response to manipulations relevant to the progression or treatment of such diseases. Additionally, such genes may have a temporally modulated expression, increased or decreased at different stages of tissue or organism development. A differentially expressed gene may also have its expression modulated under control versus experimental conditions. In addition, the human phosphatidylinositol-specific phospholipase C-like enzyme gene or gene product may itself be tested for differential expression.

[0150] The degree to which expression differs in a normal versus a diseased state need only be large enough to be visualized via standard characterization techniques such as differential display techniques. Other such standard characterization techniques by which expression differences may be visualized include but are not limited to, quantitative RT (reverse transcriptase), PCR, and Northern analysis.

[0151] Identification of Differentially Expressed Genes

[0152] To identify differentially expressed genes total RNA or, preferably, mRNA is isolated from tissues of interest. For example, RNA samples are obtained from tissues of experimental subjects and from corresponding tissues of control subjects. Any RNA isolation technique that does not select against the isolation of mRNA may be utilized for the purification of such RNA samples. See, for example, Ausubel et al., ed., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. New York, 1987-1993. Large numbers of tissue samples may readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski, U.S. Pat. No. 4,843,155.

[0153] Transcripts within the collected RNA samples that represent RNA produced by differentially expressed genes are identified by methods well known to those of skill in the art. They include, for example, differential screening (Tedder et al., Proc. Natl. Acad. Sci. U.S.A. 85, 208-12, 1988), subtractive hybridization (Hedrick et al., Nature 308, 149-53; Lee et al., Proc. Natl. Acad. Sci. U.S.A. 88, 2825, 1984), and differential display (Liang & Pardee, Science 257, 967-71, 1992; U.S. Pat. No. 5,262,311), and microarrays.

[0154] The differential expression information may itself suggest relevant methods for the treatment of disorders involving the human phosphatidylinositol-specific phospholipase C-like enzyme. For example, treatment may include a modulation of expression of the differentially expressed genes and/or the gene encoding the human phosphatidylinositol-specific phospholipase C-like enzyme. The differential expression information may indicate whether the expression or activity of the differentially expressed gene or gene product or the human phosphatidylinositol-specific phospholipase C-like enzyme gene or gene product are up-regulated or down-regulated.

[0155] Screening Methods

[0156] The invention provides assays for screening test compounds which bind to or modulate the activity of a PI-PLC-like enzyme polypeptide or a PI-PLC-like enzyme polynucleotide. A test compound preferably binds to a PI-PLC-like enzyme polypeptide or polynucleotide. More preferably, a test compound decreases or increases PI-PLC-like by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.

[0157] Test Compounds

[0158] Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the “one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997.

[0159] Methods for the synthesis of molecular libraries are well known in the art (see, for example, DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994). Libraries of compounds can be presented in solution (see, e.g., Houghten, BioTechniques 13, 412-421, 1992), or on beads (Lam, Nature 354, 82-84, 1991), chips (Fodor, Nature 364, 555-556, 1993), bacteria or spores (Ladner, U.S. Pat. No. 5,223,409), plasmids (Cull et al., Proc. Natl. Acad. Sci. U.S.A. 89, 1865-1869, 1992), or phage (Scott & Smith, Science 249, 386-390, 1990; Devlin, Science 249, 404-406, 1990); Cwirla et al., Proc. Natl. Acad. Sci. 97, 6378-6382, 1990; Felici, J. Mol. Biol. 222, 301-310, 1991; and Ladner, U.S. Pat. No. 5,223,409).

[0160] High Throughput Screening

[0161] Test compounds can be screened for the ability to bind to PI-PLC-like enzyme polypeptides or polynucleotides or to affect PI-PLC-like enzyme activity or PI-PLC-like enzyme gene expression using high throughput screening. Using high throughput screening, many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened. The most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 μl. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.

[0162] Alternatively, “free format assays,” or assays that have no physical barrier between samples, can be used. For example, an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al., Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994). The cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose. The combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.

[0163] Another example of a free format assay is described by Chelsky, “Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches,” reported at the First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995). Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel. Thereafter, beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.

[0164] Yet another example is described by Salmon et al., Molecular Diversity 2, 57-63 (1996). In this example, combinatorial libraries were screened for compounds that had cytotoxic effects on cancer cells growing in agar.

[0165] Another high throughput screening method is described in Beutel et al., U.S. Pat. No. 5,976,813. In this method, test samples are placed in a porous matrix. One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support. When samples are introduced to the porous matrix they diffuse sufficiently slowly, such that the assays can be performed without the test samples running together.

[0166] Binding Assays

[0167] For binding assays, the test compound is preferably a small molecule which binds to and occupies, for example, the ATP/GTP binding site of the enzyme or the active site of the PI-PLC-like enzyme polypeptide, such that normal biological activity is prevented. Examples of such small molecules include, but are not limited to, small peptides or peptide-like molecules.

[0168] In binding assays, either the test compound or the PI-PLC-like enzyme polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound which is bound to the PI-PLC-like enzyme polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.

[0169] Alternatively, binding of a test compound to a PI-PLC-like enzyme polypeptide can be determined without labeling either of the interactants. For example, a microphysiometer can be used to detect binding of a test compound with a PI-PLC-like enzyme polypeptide. A microphysiometer (e.g., Cytosensor™) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and a PI-PLC-like enzyme polypeptide (McConnell et al., Science 257, 1906-1912, 1992).

[0170] Determining the ability of a test compound to bind to a PI-PLC-like enzyme polypeptide also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal. Chem. 63, 2338-2345, 1991, and Szabo et al., Curr. Opin. Struct. Biol. 5, 699-705, 1995). BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore™). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

[0171] In yet another aspect of the invention, a PI-PLC-like enzyme polypeptide can be used as a “bait protein” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al., Cell 72, 223-232, 1993; Madura et al., J. Biol. Chem. 268, 12046-12054, 1993; Bartel et al., BioTechniques 14, 920-924, 1993; Iwabuchi et al., Oncogene 8, 1693-1696, 1993; and Brent WO94/10300), to identify other proteins which bind to or interact with the PI-PLC-like enzyme polypeptide and modulate its activity.

[0172] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. For example, in one construct, polynucleotide encoding a PI-PLC-like enzyme polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct a DNA sequence that encodes an unidentified protein (“prey” or “sample”) can be fused to a polynucleotide that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact in vivo to form an protein-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein which interacts with the PI-PLC-like enzyme polypeptide.

[0173] It may be desirable to immobilize either the PI-PLC-like enzyme polypeptide (or polynucleotide) or the test compound to facilitate separation of bound from unbound forms of one or both of the interactants, as well as to accommodate automation of the assay. Thus, either the PI-PLC-like enzyme polypeptide (or polynucleotide) or the test compound can be bound to a solid support. Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads). Any method known in the art can be used to attach the enzyme polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support. Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to a v enzyme polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.

[0174] In one embodiment, the PI-PLC-like enzyme polypeptide is a fusion protein comprising a domain that allows the PI-PLC-like enzyme polypeptide to be bound to a solid support. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed PI-PLC-like enzyme polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.

[0175] Other techniques for immobilizing proteins or polynucleotides on a solid support also can be used in the screening assays of the invention. For example, either a PI-PLC-like enzyme polypeptide (or polynucleotide) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated PI-PLC-like enzyme polypeptides (or polynucleotides) or test compounds can be prepared from biotin-NHS(N-hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.) and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies which specifically bind to a PI-PLC-like enzyme polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the ATP/GTP binding site or the active site of the PI-PLC-like enzyme polypeptide, can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.

[0176] Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies which specifically bind to the PI-PLC-like enzyme polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the PI-PLC-like enzyme polypeptide, and SDS gel electrophoresis under non-reducing conditions.

[0177] Screening for test compounds which bind to a PI-PLC-like enzyme polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a PI-PLC-like enzyme polypeptide or polynucleotide can be used in a cell-based assay system. A PI-PLC-like enzyme polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to a PI-PLC-like enzyme polypeptide or polynucleotide is determined as described above.

[0178] Enzyme Assays

[0179] Test compounds can be tested for the ability to increase or decrease the phospholipase C activity of a human PI-PLC-like enzyme polypeptide. Phospholipase C activity can be measured, for example, as described in the specific examples, below. Enzyme assays can be carried out after contacting either a purified PI-PLC-like enzyme polypeptide, a cell membrane preparation, or an intact cell with a test compound. A test compound which decreases a phospholipase C activity of a PI-PLC-like enzyme polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for decreasing PI-PLC-like enzyme activity. A test compound which increases a phospholipase C activity of a human PI-PLC-like enzyme polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for increasing human PI-PLC-like enzyme activity.

[0180] Gene Expression

[0181] In another embodiment, test compounds which increase or decrease PI-PLC-like enzyme gene expression are identified. A PI-PLC-like enzyme polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the v enzyme polynucleotide is determined. The level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound. The test compound can then be identified as a modulator of expression based on this comparison. For example, when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.

[0182] The level of v enzyme mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used. The presence of polypeptide products of a PI-PLC-like enzyme polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry. Alternatively, polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a PI-PLC-like enzyme polypeptide.

[0183] Such screening can be carried out either in a cell-free assay system or in an intact cell. Any cell which expresses a PI-PLC-like enzyme polynucleotide can be used in a cell-based assay system. The PI-PLC-like enzyme polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used.

[0184] Pharmaceutical Compositions

[0185] The invention also provides pharmaceutical compositions which can be administered to a patient to achieve a therapeutic effect. Pharmaceutical compositions of the invention can comprise, for example, a PI-PLC-like enzyme polypeptide, PI-PLC-like enzyme polynucleotide, ribozymes or antisense oligonucleotides, antibodies which specifically bind to a PI-PLC-like enzyme polypeptide, or mimetics, activators, or inhibitors of a PI-PLC-like enzyme polypeptide activity. The compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.

[0186] In addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Pharmaceutical compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means. Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

[0187] Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

[0188] Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

[0189] Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.

[0190] Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic amino polymers also can be used for delivery. Optionally, the suspension also can contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0191] The pharmaceutical compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. The pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use. Further details on techniques for formulation and administration can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Maack Publishing Co., Easton, Pa.). After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.

[0192] Therapeutic Indications and Methods

[0193] Human PI-PLC-like enzyme can be regulated to treat cancer. Cancer is a disease fundamentally caused by oncogenic cellular transformation. There are several hallmarks of transformed cells that distinguish them from their normal counterparts and underlie the pathophysiology of cancer. These include uncontrolled cellular proliferation, unresponsiveness to normal death-inducing signals (immortalization), increased cellular motility and invasiveness, increased ability to recruit blood supply through induction of new blood vessel formation (angiogenesis), genetic instability, and dysregulated gene expression. Various combinations of these aberrant physiologies, along with the acquisition of drug-resistance frequently lead to an intractable disease state in which organ failure and patient death ultimately ensue.

[0194] Most standard cancer therapies target cellular proliferation and rely on the differential proliferative capacities between transformed and normal cells for their efficacy. This approach is hindered by the facts that several important normal cell types are also highly proliferative and that cancer cells frequently become resistant to these agents. Thus, the therapeutic indices for traditional anti-cancer therapies rarely exceed 2.0.

[0195] The advent of genomics-driven molecular target identification has opened up the possibility of identifying new cancer-specific targets for therapeutic intervention that will provide safer, more effective treatments for cancer patients. Thus, newly discovered tumor-associated genes and their products can be tested for their role(s) in disease and used as tools to discover and develop innovative therapies. Genes playing important roles in any of the physiological processes outlined above can be characterized as cancer targets.

[0196] Genes or gene fragments identified through genomics can readily be expressed in one or more heterologous expression systems to produce functional recombinant proteins. These proteins are characterized in vitro for their biochemical properties and then used as tools in high-throughput molecular screening programs to identify chemical modulators of their biochemical activities. Activators and/or inhibitors of target protein activity can be identified in this manner and subsequently tested in cellular and in vivo disease models for anti-cancer activity. Optimization of lead compounds with iterative testing in biological models and detailed pharmacokinetic and toxicological analyses form the basis for drug development and subsequent testing in humans.

[0197] Human PI-PLC-like enzyme also can be regulated to treat CNS diseases and chronic obstructive pulmonary disease. CNS disorders which can be treated include brain injuries, cerebrovascular diseases and their consequences, Parkinson's disease, corticobasal degeneration, motor neuron disease, dementia, including ALS, multiple sclerosis, traumatic brain injury, stroke, post-stroke, post-traumatic brain injury, and small-vessel cerebrovascular disease. Dementias, such as Alzheimer's disease, vascular dementia, dementia with Lewy bodies, frontotemporal dementia and Parkinsonism linked to chromosome 17, frontotemporal dementias, including Pick's disease, progressive nuclear palsy, corticobasal degeneration, Huntington's disease, thalamic degeneration, Creutzfeld-Jakob dementia, HIV dementia, schizophrenia with dementia, and Korsakoff's psychosis also can be treated. Similarly, it is possible to treat cognitive-related disorders, such as mild cognitive impairment, age-associated memory impairment, age-related cognitive decline, vascular cognitive impairment, attention deficit disorders, attention deficit hyperactivity disorders, and memory disturbances in children with learning disabilities, by regulating the activity of human PI-PLC-like enzyme.

[0198] Pain that is associated with CNS disorders also can be treated by regulating the activity of human phosphatidylinositol-specific phospholipase C-like enzyme. Pain which can be treated includes that associated with central nervous system disorders, such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascular lesions in the brain and spinal cord (e.g., infarct, hemorrhage, vascular malformation). Non-central neuropathic pain includes that associated with post mastectomy pain, reflex sympathetic dystrophy (RSD), trigeminal neuralgiaradioculopathy, post-surgical pain, HIV/AIDS related pain, cancer pain, metabolic neuropathies (e.g., diabetic neuropathy, vasculitic neuropathy secondary to connective tissue disease), paraneoplastic polyneuropathy associated, for example, with carcinoma of lung, or leukemia, or lymphoma, or carcinoma of prostate, colon or stomach, trigeminal neuralgia, cranial neuralgias, and post-herpetic neuralgia. Pain associated with cancer and cancer treatment also can be treated, as can headache pain (for example, migraine with aura, migraine without aura, and other migraine disorders), episodic and chronic tension-type headache, tension-type like headache, cluster headache, and chronic paroxysmal hemicrania.

[0199] Human phosphatidylinositol-specific phospholipase C-like enzyme also can be regulated to treat COPD. Chronic obstructive pulmonary (or airways) disease (COPD) is a condition defined physiologically as airflow obstruction that generally results from a mixture of emphysema and peripheral airway obstruction due to chronic bronchitis (Senior & Shapiro, Pulmonary Diseases and Disorders, 3d ed., New York, McGraw-Hill, 1998, pp. 659-681, 1998; Barnes, Chest 117, 10S-14S, 2000). Emphysema is characterized by destruction of alveolar walls leading to abnormal enlargement of the air spaces of the lung. Chronic bronchitis is defined clinically as the presence of chronic productive cough for three months in each of two successive years. In COPD, airflow obstruction is usually progressive and is only partially reversible. By far the most important risk factor for development of COPD is cigarette smoking, although the disease does occur in non-smokers.

[0200] Chronic inflammation of the airways is a key pathological feature of COPD (Senior & Shapiro, 1998). The inflammatory cell population comprises increased numbers of macrophages, neutrophils, and CD8⁺ lymphocyes. Inhaled irritants, such as cigarette smoke, activate macrophages which are resident in the respiratory tract, as well as epithelial cells leading to release of chemokines (e.g., interleukin-8) and other chemotactic factors. These chemotactic factors act to increase the neutrophil/monocyte trafficking from the blood into the lung tissue and airways. Neutrophils and monocytes recruited into the airways can release a variety of potentially damaging mediators such as proteolytic enzymes and reactive oxygen species. Matrix degradation and emphysema, along with airway wall thickening, surfactant dysfunction, and mucus hypersecretion, all are potential sequelae of this inflammatory response that lead to impaired airflow and gas exchange.

[0201] PI-PLC isozymes are critical components of the transduction machinery responsible for cell activation in response to occupancy of G-protein coupled receptors. Jiang et al., Proc. Natl. Acad. Sci. 94, 7971-75, 1997; Sternweis & Smrcka, Ciba Found. Symp. 176, 96-11, 1993. Hydrolysis of membrane inositol containing phospholipids by specific PLC isozymes produces inositol phosphates that can act as calcium releasing second messengers (e.g., inositol 1,4,5-trisphosphate), and the protein kinase C activator, diacylglycerol.

[0202] PLCs currently are classified into b, g and d families. Subtypes within these families have been identified. For example, the b family consists of 4 subtypes. There is clear evidence of differential tissue distribution of these isoforms and, importantly, differential activation by G-protein subunits. Studies on the inflammatory response in knock-out mice lacking the gene for PLC b-2 have shown selective abrogation of certain features of the inflammatory response. PI-PLC subtypes are therefore attractive therapeutic targets for the inhibition of the inflammatory response in COPD.

[0203] This invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or a PI-PLC-like enzyme polypeptide binding molecule) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

[0204] A reagent which affects PI-PLC-like enzyme activity can be administered to a human cell, either in vitro or in vivo, to reduce PI-PLC-like enzyme activity. The reagent preferably binds to an expression product of a human PI-PLC-like enzyme gene. If the expression product is a protein, the reagent is preferably an antibody. For treatment of human cells ex vivo, an antibody can be added to a preparation of stem cells which have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.

[0205] In one embodiment, the reagent is delivered using a liposome. Preferably, the liposome is stable in the animal into which it has been administered for at least about 30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours. A liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human. Preferably, the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung, liver, spleen, heart brain, lymph nodes, and skin.

[0206] A liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell. Preferably, the transfection efficiency of a liposome is about 0.5 μg of DNA per 16 nmole of liposome delivered to about 10⁶ cells, more preferably about 1.0 μg of DNA per 16 nmole of liposome delivered to about 10⁶ cells, and even more preferably about 2.0 μg of DNA per 16 nmol of liposome delivered to about 10⁶ cells. Preferably, a liposome is between about 100 and 500 nm, more preferably between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.

[0207] Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol. Optionally, a liposome comprises a compound capable of targeting the liposome to a particular cell type, such as a cell-specific ligand exposed on the outer surface of the liposome.

[0208] Complexing a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods which are standard in the art (see, for example, U.S. Pat. No. 5,705,151). Preferably, from about 0.1 μg to about 10 μg of polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 μg to about 5 μg of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 μg of polynucleotides is combined with about 8 nmol liposomes.

[0209] In another embodiment, antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery. Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al. Trends in Biotechnol. 11, 202-05 (1993); Chiou et al., GENE THERAPEUTICS: METHODS AND APPLICATfONS OF DIRECT GENE TRANSFER (J. A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu et al., J. Biol. Chem. 269, 542-46 (1994); Zenke et al., Proc. Natl. Acad. Sci. U.S.A. 87, 3655-59 (1990); Wu et al., J. Biol. Chem. 266, 338-42 (1991).

[0210] Determination of a Therapeutically Effective Dose

[0211] The determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient which increases or decreases PI-PLC-like enzyme activity relative to the PI-PLC-like enzyme activity which occurs in the absence of the therapeutically effective dose.

[0212] For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

[0213] Therapeutic efficacy and toxicity, e.g., ED₅₀ (the dose therapeutically effective in 50% of the population) and LD₅₀ (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD₅₀/ED₅₀.

[0214] Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

[0215] The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.

[0216] Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

[0217] If the reagent is a single-chain antibody, polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well-established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, “gene gun,” and DEAE- or calcium phosphate-mediated transfection.

[0218] Effective in vivo dosages of an antibody are in the range of about 5 μg to about 50 μg/kg, about 50 μg to about 5 mg/kg, about 100 μg to about 500 μg/kg of patient body weight, and about 200 to about 250 μg/kg of patient body weight. For administration of polynucleotides encoding single-chain antibodies, effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA.

[0219] If the expression product is mRNA, the reagent is preferably an antisense oligonucleotide or a ribozyme. Polynucleotides which express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.

[0220] Preferably, a reagent reduces expression of a PI-PLC-like enzyme gene or the activity of a PI-PLC-like enzyme polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent. The effectiveness of the mechanism chosen to decrease the level of expression of a PI-PLC-like enzyme gene or the activity of a PI-PLC-like enzyme polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to PI-PLC-like enzyme-specific mRNA, quantitative RT-PCR, immunologic detection of a PI-PLC-like enzyme polypeptide, or measurement of PI-PLC-like enzyme activity.

[0221] In any of the embodiments described above, any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

[0222] Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

[0223] Diagnostic Methods

[0224] Human PI-PLC-like enzyme also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences which encode the enzyme. For example, differences can be determined between the cDNA or genomic sequence encoding PI-PLC-like enzyme in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.

[0225] Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method. In addition, cloned DNA segments can be employed as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags.

[0226] Genetic testing based on DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al., Science 230, 1242, 1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci. USA 85, 4397-4401, 1985). Thus, the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA. In addition to direct methods such as gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

[0227] Altered levels of a PI-PLC-like enzyme also can be detected in various tissues. Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays.

[0228] All patents and patent applications cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples which are provided for purposes of illustration only and are not intended to limit the scope of the invention.

EXAMPLE 1

[0229] Detection of PI-PLC-Like Enzyme Activity

[0230] The polynucleotide of SEQ ID NO: 1 is inserted into the expression vector pCEV4 and the expression vector pCEV4-PI-PLC-like enzyme polypeptide obtained is transfected into human embryonic kidney 293 cells. From these cells extracts are obtained and phosphatidylinositol hydrolyzing activity is measured by incubation for 10 min and 37° C. of a mixture containing 10 mM Hepes, pH 7,0, 10 mM NaCl, 4 mM MgSO4, 100 mM KCl, 0,1% deoxycholate, 50 μM cell extract, 250 μM PE containing 25,000 dpm of [3H]cell extract, 10 μM free Ca++ in the presence of 2 mM EGTA in a total volume of 100 μl. The reaction is terminated by the addition of 500 μl of chloroform, methanol, 0.1 M HCl (200:100:0,6) followed by the addition of 150 μl of 1 N HCl, 5 mM EGTA. A 0,2-ml aliquot of the upper aqueous phase is removed for measurement of radioactivity. Free Ca++ concentrations are calculated. It is shown that the polypeptide of SEQ ID NO: 2 has a phospholipase C-like enzyme activity.

EXAMPLE 2

[0231] Expression of Recombinant Human PI-PLC-Like Enzyme

[0232] The Pichia pastoris expression vector pPICZB (Invitrogen, San Diego, Calif.) is used to produce large quantities of recombinant human PI-PLC-like polypeptides in yeast. The PI-PLC-like enzyme-encoding DNA sequence is derived from SEQ ID NO:1. Before insertion into vector pPICZB, the DNA sequence is modified by well known methods in such a way that it contains at its 5′-end an initiation codon and at its 3′-end an enterokinase cleavage site, a His6 reporter tag and a termination codon. Moreover, at both termini recognition sequences for restriction endonucleases are added and after digestion of the multiple cloning site of pPICZ B with the corresponding restriction enzymes the modified DNA sequence is ligated into pPICZB. This expression vector is designed for inducible expression in Pichia pastoris, driven by a yeast promoter. The resulting pPICZ/md-His6 vector is used to transform the yeast.

[0233] The yeast is cultivated under usual conditions in 5 liter shake flasks and the recombinantly produced protein isolated from the culture by affinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea. The bound polypeptide is eluted with buffer, pH 3.5, and neutralized. Separation of the polypeptide from the His6 reporter tag is accomplished by site-specific proteolysis using enterokinase (Invitrogen, San Diego, Calif.) according to manufacturer's instructions. Purified human PI-PLC-like enzyme polypeptide is obtained.

EXAMPLE 3

[0234] Identification of Test Compounds that Bind to PI-PLC-Like Enzyme Polypeptides

[0235] Purified PI-PLC-like enzyme polypeptides comprising a glutathione-S-transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution. Human PI-PLC-like enzyme polypeptides comprise the amino acid sequence shown in SEQ ID NO:2. The test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.

[0236] The buffer solution containing the test compounds is washed from the wells. Binding of a test compound to a PI-PLC-like enzyme polypeptide is detected by fluorescence measurements of the contents of the wells. A test compound which increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound is not incubated is identified as a compound which binds to a PI-PLC-like enzyme polypeptide.

EXAMPLE 4

[0237] Identification of a Test Compound which Decreases PI-PLC-like Enzyme Gene Expression

[0238] A test compound is administered to a culture of human cells transfected with a PI-PLC-like enzyme expression construct and incubated at 37° C. for 10 to 45 minutes. A culture of the same type of cells which have not been transfected is incubated for the same time without the test compound to provide a negative control.

[0239] RNA is isolated from the two cultures as described in Chirgwin et al., Biochem. 18, 5294-99, 1979). Northern blots are prepared using 20 to 30 μg total RNA and hybridized with a ³²P-labeled PI-PLC-like enzyme-specific probe at 65° C. in Express-hyb (CLONTECH). The probe comprises at least 11 contiguous nucleotides selected from SEQ ID NO:1. A test compound which decreases the PI-PLC-like enzyme-specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of PI-PLC-like enzyme gene expression.

EXAMPLE 5

[0240] Identification of a Test Compound which Decreases PI-PLC-Like Enzyme Activity

[0241] A test compound is administered to a culture of human cells transfected with a PI-PLC-like enzyme expression construct and incubated at 37° C. for 10 to 45 minutes. A culture of the same type of cells which have not been transfected is incubated for the same time without the test compound to provide a negative control.

[0242] Phospholipase C activity can be measured using assays known in the art (e.g., Mullinax et al., J. Biomol. Screen. 4, 151-55, 1999; Litosch, Biochemistry 39, 7736-43, 2000). A test compound which decreases the phospholipase C activity of the PI-PLC-like enzyme relative to the phospholipase C activity in the absence of the test compound is identified as an inhibitor of PI-PLC-like enzyme activity.

EXAMPLE 6

[0243] Treatment of Cancer with a Reagent which Specifically Binds to a PI-PLC-Like Enzyme Gene Product

[0244] Synthesis of antisense PI-PLC-like enzyme oligonucleotides comprising at least 11 contiguous nucleotides selected from SEQ ID NO:1 is performed on a Pharmacia Gene Assembler series synthesizer using the phosphoramidite procedure (Uhlmann et al., Chem. Rev. 90, 534-83, 1990). Following assembly and deprotection, oligonucleotides are ethanol-precipitated twice, dried, and suspended in phosphate-buffered saline (PBS) at the desired concentration. Purity of these oligonucleotides is tested by capillary gel electrophoreses and ion exchange HPLC. Endotoxin levels in the oligonucleotide preparation are determined using the Limulus Amebocyte Assay (Bang, Biol. Bull. (Woods Hole, Mass.) 105, 361-362, 1953).

[0245] The antisense oligonucleotides are administered directly to a tumor of a patient with cancer. The size of the patient's tumor is decreased.

EXAMPLE 7

[0246] Proliferation Inhibition Assay: Antisense Oligonucleotides Suppress the Growth of Cancer Cell Lines

[0247] The cell line used for testing is the human colon cancer cell line HCT116. Cells are cultured in RPMI-1640 with 10-15% fetal calf serum at a concentration of 10,000 cells per milliliter in a volume of 0.5 ml and kept at 37° C. in a 95% air/5%CO₂ atmosphere.

[0248] Phosphorothioate oligoribonucleotides are synthesized on an Applied Biosystems Model 380B DNA synthesizer using phosphoroamidite chemistry. A sequence of 24 bases complementary to the nucleotides at position 1 to 24 of SEQ ID NO:1 is used as the test oligonucleotide. As a control, another (random) sequence is used: 5′-TCA ACT GAC TAG ATG TAC ATG GAC-3′. Following assembly and deprotection, oligonucleotides are ethanol-precipitated twice, dried, and suspended in phosphate buffered saline at the desired concentration. Purity of the oligonucleotides is tested by capillary gel electrophoresis and ion exchange HPLC. The purified oligonucleotides are added to the culture medium at a concentration of 10 μM once per day for seven days.

[0249] The addition of the test oligonucleotide for seven days results in significantly reduced expression of human serine palmitoyltransferase as determined by Western blotting. This effect is not observed with the control oligonucleotide. After 3 to 7 days, the number of cells in the cultures is counted using an automatic cell counter. The number of cells in cultures treated with the test oligonucleotide (expressed as 100%) is compared with the number of cells in cultures treated with the control oligonucleotide. The number of cells in cultures treated with the test oligonucleotide is not more than 30% of control, indicating that the inhibition of human serine palmitoyltransferase has an anti-proliferative effect on cancer cells.

EXAMPLE 8

[0250] In Vivo Testing of Compounds/Target Validation

[0251] 1. Acute Mechanistic Assays

[0252] 1.1. Reduction in Mitogenic Plasma Hormone Levels

[0253] This non-tumor assay measures the ability of a compound to reduce either the endogenous level of a circulating hormone or the level of hormone produced in response to a biologic stimulus. Rodents are administered test compound (p.o., i.p., i.v., i.m., or s.c.). At a predetermined time after administration of test compound, blood plasma is collected. Plasma is assayed for levels of the hormone of interest. If the normal circulating levels of the hormone are too low and/or variable to provide consistent results, the level of the hormone may be elevated by a pre-treatment with a biologic stimulus (i.e., LHRH may be injected i.m. into mice at a dosage of 30 ng/mouse to induce a burst of testosterone synthesis). The timing of plasma collection would be adjusted to coincide with the peak of the induced hormone response. Compound effects are compared to a vehicle-treated control group. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test. Significance is p value ≦0.05 compared to the vehicle control group.

[0254] 1.2. Hollow Fiber Mechanism of Action Assay

[0255] Hollow fibers are prepared with desired cell line(s) and implanted intraperitoneally and/or subcutaneously in rodents. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested in accordance with specific readout assay protocol, these may include assays for gene expression (bDNA, PCR, or Taqman), or a specific biochemical activity (i.e., cAMP levels. Results are analyzed by Student's t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p≦0.05 as compared to the vehicle control group.

[0256] 2. Subacute Functional in Vivo Assays

[0257] 2.1. Reduction in Mass of Hormone Dependent Tissues

[0258] This is another non-tumor assay that measures the ability of a compound to reduce the mass of a hormone dependent tissue (i.e., seminal vesicles in males and uteri in females). Rodents are administered test compound (p.o., i.p., i.v., i.m., or s.c.) according to a predetermined schedule and for a predetermined duration (i.e., 1 week). At termination of the study, animals are weighed, the target organ is excised, any fluid is expressed, and the weight of the organ is recorded. Blood plasma may also be collected. Plasma may be assayed for levels of a hormone of interest or for levels of test agent. Organ weights may be directly compared or they may be normalized for the body weight of the animal. Compound effects are compared to a vehicle-treated control group. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test. Significance is p value ≦0.05 compared to the vehicle control group.

[0259] Hollows Fiber Proliferation Assay

[0260] Hollow fibers are prepared with desired cell line(s) and implanted intraperitoneally and/or subcutaneously in rodents. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested in accordance with specific readout assay protocol. Cell proliferation is determined by measuring a marker of cell number (i.e., MTT or LDH). The cell number and change in cell number from the starting inoculum are analyzed by Student's t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p≦0.05 as compared to the vehicle control group.

[0261] 2.2. Anti-Angiogenesis Models

[0262] 2.2.1. Corneal Angiogenesis

[0263] Hydron pellets with or without growth factors or cells are implanted into a micropocket surgically created in the rodent cornea. Compound administration may be systemic or local (compound mixed with growth factors in the hydron pellet). Corneas are harvested at 7 days post implantation immediately following intracardiac infusion of colloidal carbon and are fixed in 10% formalin. Readout is qualitative scoring and/or image analysis. Qualitative scores are compared by Rank Sum test. Image analysis data is evaluated by measuring the area of neovascularization (in pixels) and group averages are compared by Student's t-test (2 tail). Significance is p≦0.05 as compared to the growth factor or cells only group.

[0264] 2.2.2. Matrigel Angiogenesis

[0265] Matrigel, containing cells or growth factors, is injected subcutaneously. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Matrigel plugs are harvested at predetermined time point(s) and prepared for readout. Readout is an ELISA-based assay for hemoglobin concentration and/or histological examination (i.e. vessel count, special staining for endothelial surface markers: CD31, factor-8). Readouts are analyzed by Student's t-test, after the variance between groups is compared by an F-test, with significance determined at p≦0.05 as compared to the vehicle control group.

[0266] 3. Primary Antitumor Efficacy

[0267] 3.1. Early Therapy Models

[0268] 3.1.1. Subcutaneous Tumor

[0269] Tumor cells or fragments are implanted subcutaneously on Day 0. Vehicle and/or compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule starting at a time, usually on Day 1, prior to the ability to measure the tumor burden. Body weights and tumor measurements are recorded 2-3 times weekly. Mean net body and tumor weights are calculated for each data collection day. Antitumor efficacy may be initially determined by comparing the size of treated (T) and control (C) tumors on a given day by a Student's t-test, after the variance between groups is compared by an F-test, with significance determined at p≦0.05. The experiment may also be continued past the end of dosing in which case tumor measurements would continue to be recorded to monitor tumor growth delay. Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan-Meier curves from the times for individual tumors to attain the evaluation size. Significance is p≦0.05.

[0270] 3.1.2. Intraperitoneal/Intracranial Tumor Models

[0271] Tumor cells are injected intraperitoneally or intracranially on Day 0. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule starting on Day 1. Observations of morbidity and/or mortality are recorded twice daily. Body weights are measured and recorded twice weekly. Morbidity/mortality data is expressed in terms of the median time of survival and the number of long-term survivors is indicated separately. Survival times are used to generate Kaplan-Meier curves. Significance is p≦0.05 by a log-rank test compared to the control group in the experiment.

[0272] 3.2. Established Disease Model

[0273] Tumor cells or fragments are implanted subcutaneously and grown to the desired size for treatment to begin. Once at the predetermined size range, mice are randomized into treatment groups. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Tumor and body weights are measured and recorded 2-3 times weekly. Mean tumor weights of all groups over days post inoculation are graphed for comparison. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p≦0.05 as compared to the control group. Tumor measurements may be recorded after dosing has stopped to monitor tumor growth delay. Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan-Meier curves from the times for individual tumors to attain the evaluation size. Significance is p value ≦0.05 compared to the vehicle control group.

[0274] 3.3. Orthotopic Disease Models

[0275] 3.3.1. Mammary Fat Pad Assay

[0276] Tumor cells or fragments, of mammary adenocarcinoma origin, are implanted directly into a surgically exposed and reflected mammary fat pad in rodents. The fat pad is placed back in its original position and the surgical site is closed. Hormones may also be administered to the rodents to support the growth of the tumors. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Tumor and body weights are measured and recorded 2-3 times weekly. Mean tumor weights of all groups over days post inoculation are graphed for comparison. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p≦0.05 as compared to the control group.

[0277] Tumor measurements may be recorded after dosing has stopped to monitor tumor growth delay. Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan-Meier curves from the times for individual tumors to attain the evaluation size. Significance is p value ≦0.05 compared to the vehicle control group. In addition, this model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ, or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p≦0.05 compared to the control group in the experiment.

[0278] 3.3.2. Intraprostatic Assay

[0279] Tumor cells or fragments, of prostatic adenocarcinoma origin, are implanted directly into a surgically exposed dorsal lobe of the prostate in rodents. The prostate is externalized through an abdominal incision so that the tumor can be implanted specifically in the dorsal lobe while verifying that the implant does not enter the seminal vesicles. The successfully inoculated prostate is replaced in the abdomen and the incisions through the abdomen and skin are closed. Hormones may also be administered to the rodents to support the growth of the tumors. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected. The size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p≦0.05 as compared to the control group. This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the lungs), or measuring the target organ weight (i.e., the regional lymph nodes). The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p≦0.05 compared to the control group in the experiment.

[0280] 3.3.3. Intrabronchial Assay

[0281] Tumor cells of pulmonary origin may be implanted intrabronchially by making an incision through the skin and exposing the trachea. The trachea is pierced with the beveled end of a 25 gauge needle and the tumor cells are inoculated into the main bronchus using a flat-ended 27 gauge needle with a 90° bend. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected. The size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p≦0.05 as compared to the control group. This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the contralateral lung), or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p≦0.05 compared to the control group in the experiment.

[0282] 3.3.4. Intracecal Assay

[0283] Tumor cells of gastrointestinal origin may be implanted intracecally by making an abdominal incision through the skin and externalizing the intestine. Tumor cells are inoculated into the cecal wall without penetrating the lumen of the intestine using a 27 or 30 gauge needle. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected. The size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p≦0.05 as compared to the control group. This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the liver), or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p≦0.05 compared to the control group in the experiment.

[0284] 4. Secondary (Metastatic) Antitumor Efficacy

[0285] 4.1. Spontaneous Metastasis

[0286] Tumor cells are inoculated s.c. and the tumors allowed to grow to a predetermined range for spontaneous metastasis studies to the lung or liver. These primary tumors are then excised. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule which may include the period leading up to the excision of the primary tumor to evaluate therapies directed at inhibiting the early stages of tumor metastasis. Observations of morbidity and/or mortality are recorded daily. Body weights are measured and recorded twice weekly. Potential endpoints include survival time, numbers of visible foci per target organ, or target organ weight. When survival time is used as the endpoint the other values are not determined. Survival data is used to generate Kaplan-Meier curves. Significance is p≦0.05 by a log-rank test compared to the control group in the experiment. The mean number of visible tumor foci, as determined under a dissecting microscope, and the mean target organ weights are compared by Student's t-test after conducting an F-test, with significance determined at p≦0.05 compared to the control group in the experiment for both of these endpoints.

[0287] 4.2. Forced Metastasis

[0288] Tumor cells are injected into the tail vein, portal vein, or the left ventricle of the heart in experimental (forced) lung, liver, and bone metastasis studies, respectively. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Observations of morbidity and/or mortality are recorded daily. Body weights are measured and recorded twice weekly. Potential endpoints include survival time, numbers of visible foci per target organ, or target organ weight. When survival time is used as the endpoint the other values are not determined. Survival data is used to generate Kaplan-Meier curves. Significance is p≦0.05 by a log-rank test compared to the control group in the experiment. The mean number of visible tumor foci, as determined under a dissecting microscope, and the mean target organ weights are compared by Student's t-test after conducting an F-test, with significance at p≦0.05 compared to the vehicle control group in the experiment for both end-points.

EXAMPLE 9

[0289] In Vivo Testing of Compounds/Target Validation

[0290] 1. Pain:

[0291] Acute Pain

[0292] Acute pain is measured on a hot plate mainly in rats. Two variants of hot plate testing are used: In the classical variant animals are put on a hot surface (52 to 56° C.) and the latency time is measured until the animals show nocifensive behavior, such as stepping or foot licking. The other variant is an increasing temperature hot plate where the experimental animals are put on a surface of neutral temperature. Subsequently this surface is slowly but constantly heated until the animals begin to lick a hind paw. The temperature which is reached when hind paw licking begins is a measure for pain threshold.

[0293] Compounds are tested against a vehicle treated control group. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c., intradermal, transdermal) prior to pain testing.

[0294] Persistent Pain

[0295] Persistent pain is measured with the formalin or capsaicin test, mainly in rats. A solution of 1 to 5% formalin or 10 to 100 μg capsaicin is injected into one hind paw of the experimental animal. After formalin or capsaicin application the animals show nocifensive reactions like flinching, licking and biting of the affected paw. The number of nocifensive reactions within a time frame of up to 90 minutes is a measure for intensity of pain.

[0296] Compounds are tested against a vehicle treated control group. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c., intradermal, transdermal) prior to formalin or capsaicin administration.

[0297] Neuropathic Pain

[0298] Neuropathic pain is induced by different variants of unilateral sciatic nerve injury mainly in rats. The operation is performed under anesthesia. The first variant of sciatic nerve injury is produced by placing loosely constrictive ligatures around the common sciatic nerve. The second variant is the tight ligation of about the half of the diameter of the common sciatic nerve. In the next variant, a group of models is used in which tight ligations or transections are made of either the L5 and L6 spinal nerves, or the L% spinal nerve only. The fourth variant involves an axotomy of two of the three terminal branches of the sciatic nerve (tibial and common peroneal nerves) leaving the remaining sural nerve intact whereas the last variant comprises the axotomy of only the tibial branch leaving the sural and common nerves uninjured. Control animals are treated with a sham operation.

[0299] Postoperatively, the nerve injured animals develop a chronic mechanical allodynia, cold allodynioa, as well as a thermal hyperalgesia. Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer, IITC Inc.-Life Science Instruments, Woodland Hills, SA, USA; Electronic von Frey System, Somedic Sales AB, Hörby, Sweden). Thermal hyperalgesia is measured by means of a radiant heat source (Plantar Test, Ugo Basile, Comerio, Italy), or by means of a cold plate of 5 to 10° C. where the nocifensive reactions of the affected hind paw are counted as a measure of pain intensity. A further test for cold induced pain is the counting of nocifensive reactions, or duration of nocifensive responses after plantar administration of acetone to the affected hind limb. Chronic pain in general is assessed by registering the circadanian rhytms in activity (Surjo and Arndt, Universität zu Köln, Cologne, Germany), and by scoring differences in gait (foot print patterns; FOOTPRINTS program, Klapdor et al., 1997. A low cost method to analyse footprint patterns. J. Neurosci. Methods 75, 49-54).

[0300] Compounds are tested against sham operated and vehicle treated control groups. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c., intradermal, transdermal) prior to pain testing.

[0301] Inflammatory Pain

[0302] Inflammatory pain is induced mainly in rats by injection of 0.75 mg carrageenan or complete Freund's adjuvant into one hind paw. The animals develop an edema with mechanical allodynia as well as thermal hyperalgesia. Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer, IITC Inc.-Life Science Instruments, Woodland Hills, SA, USA). Thermal hyperalgesia is measured by means of a radiant heat source (Plantar Test, Ugo Basile, Comerio, Italy, Paw thermal stimulator, G. Ozaki, University of California, USA). For edema measurement two methods are being used. In the first method, the animals are sacrificed and the affected hindpaws sectioned and weighed. The second method comprises differences in paw volume by measuring water displacement in a plethysmometer (Ugo Basile, Comerio, Italy).

[0303] Compounds are tested against uninflamed as well as vehicle treated control groups. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c., intradermal, transdermal) prior to pain testing.

[0304] Diabetic Neuropathic Pain

[0305] Rats treated with a single intraperitoneal injection of 50 to 80 mg/kg streptozotocin develop a profound hyperglycemia and mechanical allodynia within 1 to 3 weeks. Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer, IITC Inc.-Life Science Instruments, Woodland Hills, SA, USA).

[0306] Compounds are tested against diabetic and non-diabetic vehicle treated control groups. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c., intradermal, transdermal) prior to pain testing.

[0307] 2. Parkinson's Disease

[0308] 6-Hydroxydopamine (6-OH-DA) Lesion

[0309] Degeneration of the dopaminergic nigrostriatal and striatopallidal pathways is the central pathological event in Parkinson's disease. This disorder has been mimicked experimentally in rats using single/sequential unilateral stereotaxic injections of 6-OH-DA into the medium forebrain bundle (MFB).

[0310] Male Wistar rats (Harlan Winkelmann, Germany), weighing 200±250 g at the beginning of the experiment, are used. The rats are maintained in a temperature- and humidity-controlled environment under a 12 h light/dark cycle with free access to food and water when not in experimental sessions. The following in vivo protocols are approved by the governmental authorities. All efforts are made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques.

[0311] Animals are administered pargyline on the day of surgery (Sigma, St. Louis, Mo., USA; 50 mg/kg i.p.) in order to inhibit metabolism of 6-OHDA by monoamine oxidase and desmethylimipramine HCl (Sigma; 25 mg/kg i.p.) in order to prevent uptake of 6-OHDA by noradrenergic terminals. Thirty minutes later the rats are anesthetized with sodium pentobarbital (50 mg/kg) and placed in a stereotaxic frame. In order to lesion the DA nigrostriatal pathway 4 μl of 0.01% ascorbic acid-saline containing 8 μg of 6-OHDA HBr (Sigma) are injected into the left medial fore-brain bundle at a rate of 1 μl/min (2.4 mm anterior, 1.49 mm lateral, −2.7 mm ventral to Bregma and the skull surface). The needle is left in place an additional 5 min to allow diffusion to occur.

[0312] Stepping Test

[0313] Forelimb akinesia is assessed three weeks following lesion placement using a modified stepping test protocol. In brief, the animals are held by the experimenter with one hand fixing the hindlimbs and slightly raising the hind part above the surface. One paw is touching the table, and is then moved slowly sideways (5 s for 1 m), first in the forehand and then in the backhand direction. The number of adjusting steps is counted for both paws in the backhand and forehand direction of movement. The sequence of testing is right paw forehand and backhand adjusting stepping, followed by left paw forehand and backhand directions. The test is repeated three times on three consecutive days, after an initial training period of three days prior to the first testing. Forehand adjusted stepping reveals no consistent differences between lesioned and healthy control animals. Analysis is therefore restricted to backhand adjusted stepping.

[0314] Balance Test

[0315] Balance adjustments following postural challenge are also measured during the stepping test sessions. The rats are held in the same position as described in the stepping test and, instead of being moved sideways, tilted by the experimenter towards the side of the paw touching the table. This manoeuvre results in loss of balance and the ability of the rats to regain balance by forelimb movements is scored on a scale ranging from 0 to 3. Score 0 is given for a normal forelimb placement. When the forelimb movement is delayed but recovery of postural balance detected, score 1 is given. Score 2 represents a clear, yet insufficient, forelimb reaction, as evidenced by muscle contraction, but lack of success in recovering balance, and score 3 is given for no reaction of movement. The test is repeated three times a day on each side for three consecutive days after an initial training period of three days prior to the first testing.

[0316] Staircase Test (Paw Reaching)

[0317] A modified version of the staircase test is used for evaluation of paw reaching behaviour three weeks following primary and secondary lesion placement. Plexiglass test boxes with a central platform and a removable staircase on each side are used. The apparatus is designed such that only the paw on the same side at each staircase can be used, thus providing a measure of independent forelimb use. For each test the animals are left in the test boxes for 15 min. The double staircase is filled with 7×3 chow pellets (Precision food pellets, formula: P, purified rodent diet, size 45 mg; Sandown Scientific) on each side. After each test the number of pellets eaten (successfully retrieved pellets) and the number of pellets taken (touched but dropped) for each paw and the success rate (pellets eaten/pellets taken) are counted separately. After three days of food deprivation (12 g per animal per day) the animals are tested for II days. Full analysis is conducted only for the last five days.

[0318] MPTP Treatment

[0319] The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP) causes degeneration of mesencephalic dopaminergic (DAergic) neurons in rodents, non-human primates, and humans and, in so doing, reproduces many of the symptoms of Parkinson's disease. MPTP leads to a marked decrease in the levels of dopamine and its metabolites, and in the number of dopaminergic terminals in the striatum as well as severe loss of the tyrosine hydroxylase (TH)-immunoreactive cell bodies in the substantia nigra, pars compacta.

[0320] In order to obtain severe and long-lasting lesions, and to reduce mortality, animals receive single injections of MPTP, and are then tested for severity of lesion 7-10 days later. Successive MPTP injections are administered on days 1, 2 and 3. Animals receive application of 4 mg/kg MPTP hydrochloride (Sigma) in saline once daily. All injections are intraperitoneal (i.p.) and the MPTP stock solution is frozen between injections. Animals are decapitated on day 11.

[0321] Immunohistology

[0322] At the completion of behavioral experiments, all animals are anaesthetized with 3 ml thiopental (1 g/40 ml i.p., Tyrol Pharma). The mice are perfused transcardially with 0.01 M PBS (pH 7.4) for 2 min, followed by 4% paraformaldehyde (Merck) in PBS for 15 min. The brains are removed and placed in 4% paraformaldehyde for 24 h at 4° C. For dehydration they are then transferred to a 20% sucrose (Merck) solution in 0.1 M PBS at 4° C. until they sink. The brains are frozen in methylbutan at −20° C. for 2 min and stored at −70° C. Using a sledge microtome (mod. 3800-Frigocut, Leica), 25 μm sections are taken from the genu of the corpus callosum (AP 1.7 mm) to the hippocampus (AP 21.8 mm) and from AP 24.16 to AP 26.72. Forty-six sections are cut and stored in assorters in 0.25 M Tris buffer (pH 7.4) for immunohistochemistry.

[0323] A series of sections is processed for free-floating tyrosine hydroxylase (TH) immunohistochemistry. Following three rinses in 0.1 M PBS, endogenous peroxidase activity is quenched for 10 min in 0.3% H₂O₂±PBS. After rinsing in PBS, sections are preincubated in 10% normal bovine serum (Sigma) for 5 min as blocking agent and transferred to either primary anti-rat TH rabbit antiserum (dilution 1:2000).

[0324] Following overnight incubation at room temperature, sections for TH immunoreactivity are rinsed in PBS (2×10 min) and incubated in biotinylated anti-rabbit immunoglobulin G raised in goat (dilution 1:200) (Vector) for 90 min, rinsed repeatedly and transferred to Vectastain ABC (Vector) solution for 1 h. 3,3′-Diaminobenzidine tetrahydrochloride (DAB; Sigma) in 0.1 M PBS, supplemented with 0.005% H₂O₂, serves as chromogen in the subsequent visualization reaction. Sections are mounted on to gelatin-coated slides, left to dry overnight, counter-stained with hematoxylin dehydrated in ascending alcohol concentrations and cleared in butylacetate. Coverslips are mounted on entellano.

[0325] Rotarod Test

[0326] We use a modification of the procedure described by Rozas and Labandeira-Garcia (1997), with a CR-1 Rotamex system (Columbus Instruments, Columbus, Ohio) comprising an IBM-compatible personal computer, a CIO-24 data acquisition card, a control unit, and a four-lane rotarod unit. The rotarod unit consists of a rotating spindle (diameter 7.3 cm) and individual compartments for each mouse. The system software allows preprogramming of session protocols with varying rotational speeds (0-80 rpm). Infrared beams are used to detect when a mouse has fallen onto the base grid beneath the rotarod. The system logs the fall as the end of the experiment for that mouse, and the total time on the rotarod, as well as the time of the fall and all the set-up parameters, are recorded. The system also allows a weak current to be passed through the base grid, to aid training.

[0327] 3. Dementia

[0328] The Object Recognition Task

[0329] The object recognition task has been designed to assess the effects of experimental manipulations on the cognitive performance of rodents. A rat is placed in an open field, in which two identical objects are present. The rats inspects both objects during the first trial of the object recognition task. In a second trial, after a retention interval of for example 24 hours, one of the two objects used in the first trial, the ‘familiar’ object, and a novel object are placed in the open field. The inspection time at each of the objects is registered. The basic measures in the OR task is the time spent by a rat exploring the two object the second trial. Good retention is reflected by higher explortation times towards the novel than the ‘familiar’ object.

[0330] Administration of the putative cognition enhancer prior to the first trial predominantly allows assessment of the effects on acquisition, and eventually on consolidation processes. Administration of the testing compound after the first trial allows to assess the effects on consolidation processes, whereas administration before the second trial allows to measure effects on retrieval processes.

[0331] The Passive Avoidance Task

[0332] The passive avoidance task assesses memory performance in rats and mice. The inhibitory avoidance apparatus consists of a two-compartment box with a light compartment and a dark compartment. The two compartments are separated by a guillotine door that can be operated by the experimenter. A threshold of 2 cm separates the two compartments when the guillotine door is raised. When the door is open, the illumination in the dark compartment is about 2 lux. The light intensity is about 500 lux at the center of the floor of the light compartment.

[0333] Two habituation sessions, one shock session, and a retention session are given, separated by inter-session intervals of 24 hours. In the habituation sessions and the retention session the rat is allowed to explore the apparatus for 300 sec. The rat is placed in the light compartment, facing the wall opposite to the guillotine door. After an accommodation period of 15 sec. the guillotine door is opened so that all parts of the apparatus can be visited freely. Rats normally avoid brightly lit areas and will enter the dark compartment within a few seconds.

[0334] In the shock session the guillotine door between the compartments is lowered as soon as the rat has entered the dark compartment with its four paws, and a scrambled 1 mA footshock is administered for 2 sec. The rat is removed from the apparatus and put back into its home cage. The procedure during the retention session is identical to that of the habituation sessions.

[0335] The step-through latency, that is the first latency of entering the dark compartment (in sec.) during the retention session is an index of the memory performance of the animal; the longer the latency to enter the dark compartment, the better the retention is. A testing compound in given half an hour before the shock session, together with 1 mg*kg⁻¹ scopolamine. Scopolamine impairs the memory performance during the retention session 24 hours later. If the test compound increases the enter latency compared with the scopolamine-treated controls, is is likely to possess cognition enhancing potential.

[0336] The Morris Water Escape Task

[0337] The Morris water escape task measures spatial orientation learning in rodents. It is a test system that has extensively been used to investigate the effects of putative therapeutic on the cognitive functions of rats and mice. The performance of an animal is assessed in a circular water tank with an escape platform that is submerged about 1 cm below the surface of the water. The escape platform is not visible for an animal swimming in the water tank. Abundant extra-maze cues are provided by the furniture in the room, including desks, computer equipment, a second water tank, the presence of the experimenter, and by a radio on a shelf that is playing softly.

[0338] The animals receive four trials during five daily acquisition sessions. A trial is started by placing an animal into the pool, facing the wall of the tank. Each of four starting positions in the quadrants north, east, south, and west is used once in a series of four trials; their order is randomized. The escape platform is always in the same position. A trial is terminated as soon as the animal had climbs onto the escape platform or when 90 seconds have elapsed, whichever event occurs first. The animal is allowed to stay on the platform for 30 seconds. Then it is taken from the platform and the next trial is started. If an animal did not find the platform within 90 seconds it is put on the platform by the experimenter and is allowed to stay there for 30 seconds. After the fourth trial of the fifth daily session, an additional trial is given as a probe trial: the platform is removed, and the time the animal spends in the four quadrants is measured for 30 or 60 seconds. In the probe trial, all animals start from the same start position, opposite to the quadrant where the escape platform had been positioned during acquisition.

[0339] Four different measures are taken to evaluate the performance of an animal during acquisition training: escape latency, traveled distance, distance to platform, and swimming speed. The following measures are evaluated for the probe trial: time (s) in quadrants and traveled distance (cm) in the four quadrants. The probe trial provides additional information about how well an animal learned the position of the escape platform. If an animal spends more time and swims a longer distance in the quadrant where the platform had been positioned during the acquisition sessions than in any other quadrant, one concludes that the platform position has been learned well.

[0340] In order to assess the effects of putative cognition enhancing compounds, rats or mice with specific brain lesions which impair cognitive functions, or animals treated with compounds such as scopolamine or MK-801, which interfere with normal leaning, or aged animals which suffer from cognitive deficits, are used.

[0341] The T-Maze Spontaneous Alternation Task

[0342] The T-maze spontaneous alternation task (TeMCAT) assesses the spatial memory performance in mice. The start arm and the two goal arms of the T-maze are provided with guillotine doors which can be operated manually by the experimenter. A mouse is put into the start arm at the beginning of training. The guillotine door is closed. In the first trial, the ‘forced trial’, either the left or right goal arm is blocked by lowering the guillotine door. After the mouse has been released from the start arm, it will negotiate the maze, eventually enter the open goal arm, and return to the start position, where it will be confined for 5 seconds, by lowering the guillotine door. Then, the animal can choose freely between the left and right goal arm (all guillotine-doors opened) during 14 ‘free choice’ trials. As soon a the mouse has entered one goal arm, the other one is closed. The mouse eventually returns to the start arm and is free to visit whichever goalarm it wants after having been confined to the start arm for 5 seconds. After completion of 14 free choice trials in one session, the animal is removed from the maze. During training, the animal is never handled.

[0343] The percent alternations out of 14 trials is calculated. This percentage and the total time needed to complete the first forced trial and the subsequent 14 free choice trials (in s) is analysed. Cognitive deficits are usually induced by an injection of scopolamine, 30 min before the start of the training session. Scopolamine reduced the per-cent alternations to chance level, or below. A cognition enhancer, which is always administered before the training session, will at least partially, antagonize the scopolamine-induced reduction in the spontaneous alternation rate.

EXAMPLE 10

[0344] Tissue-Specific Expression of Phosphatidylinositol-Specific Phospholipase C-Like Enzyme

[0345] As a first step to establishing a role for phosphatidylinositol-specific phospholipase C-like enzyme in the pathogenesis of COPD, expression profiling of the gene was done using real-time quantitative PCR with RNA samples from human respiratory tissues and inflammatory cells relevant to COPD. The panel consisted of total RNA samples lung (adult and fetal), trachea, freshly isolated alveolar type II cells, cultured human bronchial epithelial cells, cultured small airway epithelial cells, cultured bronchial sooth muscle cells, cultured H441 cells (Clara-like), freshly isolated neutrophils and monocytes, and cultured monocytes (macrophage-like). Expression of phosphatidylinositol-specific phospholipase C-like enzyme also was evaluated in a range of human tissues using total RNA panels obtained from Clontech Laboratories, UK, Ltd. The tissues were adrenal gland, bone marrow, brain, colon, heart, kidney, liver, lung, mammary gland, pancreas, prostate, salivary gland, skeletal muscle, small intestine, spleen, stomach, testis, thymus, trachea, thyroid, and uterus.

[0346] Real-time quantitative PCR. Expression profiling of the target gene was performed using real-time quantitative PCR, a development of the kinetic analysis of PCR first described in Higuchi et al., BioTechnology 10, 413-17, 1992, and Higuchi et al., BioTechnology, 11, 1026-30, 1993. The principle is that at any given cycle within the exponential phase of PCR, the amount of product is proportional to the initial number of template copies.

[0347] PCR amplification is performed in the presence of an oligonucleotide probe (TaqMan probe) that is complementary to the target sequence and labeled with a fluorescent reporter dye and a quencher dye. During the extension phase of PCR, the probe is cleaved by the 5′-3′ endonuclease activity of Taq DNA polymerase, releasing the fluorophore from the effect of the quenching dye (Holland et al., Proc. Natl. Acad. Sci. U.S.A. 88, 7276-80, 1991). Because the fluorescence emission increases in direct proportion to the amount of the specific amplified product, the exponential growth phase of PCR product can be detected and used to determine the initial template concentration (Heid et al., Genome Res. 6, 986-94, 1996, and Gibson et al., Genome Res. 6, 995-1001, 1996).

[0348] Real-time quantitative PCR was done using an ABI Prism 7700 Sequence Detector. The C_(T) value generated for each reaction was used to determine the initial template concentration (copy number) by interpolation from a universal standard curve. The level of expression of the target gene in each sample was calculated relative to the sample with the lowest expression of the gene.

[0349] RNA extraction and cDNA preparation. Total RNA from each of the respiratory tissues and inflammatory cell types listed above were isolated using Qiagen's RNeasy system according to the manufacturer's protocol (Crawley, West Sussex, UK). The concentration of purified RNA was determined using a RiboGreen RNA quantitation kit (Molecular Probes Europe, The Netherlands). For the preparation of cDNA, 1 μg of total RNA was reverse transcribed in a final volume of 20 μl, using 200 U of SUPERSCRJPT™ RNase H-Reverse Transcriptase (Life Technologies, Paisley, UK), 10 mM dithiothreitol, 0.5 mM of each dNTP and 5 μM random hexamers (Applied Biosystems, Warrington, Cheshire, UK) according to the manufacturer's protocol.

[0350] TaqMan quantitative analysis. Specific primers and probe were designed according to the recommendations of PE Applied Biosystems. The probe was labeled at the 5′ end with FAM (6-carboxyfluorescein). Quantification PCR was performed with 5 ng of reverse transcribed RNA from each sample. Each determination is done in duplicate.

[0351] The assay reaction mix was as follows: 1× final TaqMan Universal PCR Master Mix (from 2× stock) (PE Applied Biosystems, CA); 900 nM forward primer; 900 nM reverse primer; 200 nM probe; 5 ng cDNA; and water to 25 μl.

[0352] Each of the following steps were carried out once: pre PCR, 2 minutes at 50° C., and 10 minutes at 95° C. The following steps are carried out 40 times: denaturation, 15 seconds at 95° C., annealing/extension, 1 minute at 60° C.

[0353] All experiments were performed using an ABI Prism 7700 Sequence Detector (PE Applied Biosystems, CA). At the end of the run, fluorescence data acquired during PCR were processed as described in the ABI Prism 7700 user's manual to achieve better background subtraction as well as signal linearity with the starting target quantity.

[0354] Tables 1 and 2 show the results of expression profiling for phosphatidylinositol-specific phospholipase C-like enzyme using the indicated cell and tissue samples. For Table 1, the cells are defined as follows: HBEC, cultured human bronchial epithelial cells; H441, a Clara-like cell line; SAE, cultured small airway epithelial cells; SMC, cultured airway smooth muscle cells; AII, freshly isolated human alveolar type II cells; Neut, freshly isolated circulating neutrophils; Mono, freshly isolated monocytes; and CM, cultured monocytes. Other letters identify the donor. The results are shown graphically in FIGS. 6 and 7. TABLE 1 Tissue Relative expression Lung 1374.941752 Trachea 299.1226502 HBEC 1 0 HBEC 2 17.46084672 H441 613.4021587 SMC 18.37157119 SAE 35.13025482 AII 61.06794419 Foetal lung 284.2943967 COPD Neut 1 1.012792321 COPD Neut 2 0 COPD Neut 4 1 GAP Neut 23.68924939 AEM Neut 1.100025113 AT Neut 2.927284951 KN Neut 1.052159547 SM Mono 8.144178805 DLF Mono 14.89571314 DS Mono 16.59526913 RLH CM 21.3987941 CTP CM 3.159267501

[0355] TABLE 2 Tissue Relative expression Adrenal gland 4.232071535 Bone Marrow 52.09655433 Brain 77.74974978 Colon 34.24837736 Heart 5.669163731 HL60 1.052158093 Kidney 12.00096033 Liver 5.056336866 Lung 54.12164646 Mammary gland 10.84061697 Pancreas 5.121026226 Prostate 32.13949141 Salivary gland 7.691410663 Skeletal Muscle 13.2013395 Sm Intest 1.436573993 Spleen 77.25702849 Stomach 23.24186638 Testis 31.33271295 Thymus 65.49000144 Thyroid 27.94570048 Uterus 1

EXAMPLE 11

[0356] Quantitative Expression Profiling

[0357] Expression profiling is based on a quantitative polymerase chain reaction (PCR) analysis, also called kinetic analysis, first described in Higuchi et al., 1992 and Higuchi et al., 1993. The principle is that at any given cycle within the exponential phase of PCR, the amount of product is proportional to the initial number of template copies. Using this technique, the expression levels of particular genes, which are transcribed from the chromosomes as messenger RNA (mRNA), are measured by first making a DNA copy (cDNA) of the mRNA, and then performing quantitative PCR on the cDNA, a method called quantitative reverse transcription-polymerase chain reaction (quantitative RT-PCR).

[0358] Quantitative RT-PCR analysis of RNA from different human tissues was performed to investigate the tissue distribution of PI-PLC-like enzyme mRNA. In most cases, 25 .mu.g of total RNA from various tissues (including Human Total RNA Panel I-V, Clontech Laboratories, Palo Alto, Calif., USA) was used as a template to synthesize first-strand cDNA using the SUPERSCRIPT™ First-Strand Synthesis System for RT-PCR (Life Technologies, Rockville, Md., USA). First-strand cDNA synthesis was carried out according to the manufacturer's protocol using oligo (dT) to hybridize to the 3′ poly A tails of mRNA and prime the synthesis reaction. 10 ng of the first-strand cDNA was then used as template in a polymerase chain reaction. In other cases, 10 ng of commercially available cDNAs (Human Immune System MTC Panel and Human Blood Fractions MTC Panel, Clontech Laboratories, Palo Alto, Calif., USA) were used as template in a polymerase chain reaction. The polymerase chain reaction was performed in a LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind., USA), in the presence of the DNA-binding fluorescent dye SYBR Green I which binds to the minor groove of the DNA double helix, produced only when double-stranded DNA is successfully synthesized in the reaction (Morrison et al., 1998). Upon binding to double-stranded DNA, SYBR Green I emits light that can be quantitatively measured by the LightCycler machine. The polymerase chain reaction was carried out using oligonucleotide primers PIPLCH-L4 (CGAGGACTGGATGTCGGCACT) and PIPLCH-R3 (TGACGGACCATTTCAGCACGA) and measurements of the intensity of emitted light were taken following each cycle of the reaction when the reaction had reached a temperature of 91 degrees C. Intensities of emitted light were converted into copy numbers of the gene transcript per nanogram of template cDNA by comparison with simultaneously reacted standards of known concentration.

[0359] To correct for differences in mRNA transcription levels per cell in the various tissue types, a normalization procedure was performed using similarly calculated expression levels in the various tissues of five different housekeeping genes: glyceraldehyde-3-phosphatase (G3PDH), hypoxanthine guanine phophoribosyl transferase (HPRT), beta-actin, porphobilinogen deaminase (PBGD), and beta-2-microglobulin. The level of housekeeping gene expression is considered to be relatively constant for all tissues (Adams et al., 1993, Adams et al., 1995, Liew et al., 1994) and therefore can be used as a gauge to approximate relative numbers of cells per .mu.g of total RNA used in the cDNA synthesis step. Except for the use of a slightly different set of housekeeping genes and the use of the LightCycler system to measure expression levels, the normalization procedure was essentially the same as that described in the RNA Master Blot User Manual, Appendix C (1997, Clontech Laboratories, Palo Alto, Calif., USA). In brief, expression levels of the five housekeeping genes in all tissue samples were measured in three independent reactions per gene using the LightCycler and a constant amount (25 .mu.g) of starting RNA. The calculated copy numbers for each gene, derived from comparison with simultaneously reacted standards of known concentrations, were recorded and converted into a percentage of the sum of the copy numbers of the gene in all tissue samples. Then for each tissue sample, the sum of the percentage values for each gene was calculated, and a normalization factor was calculated by dividing the sum percentage value for each tissue by the sum percentage value of one of the tissues arbitrarily selected as a standard. To normalize an experimentally obtained value for the expression of a particular gene in a tissue sample, the obtained value was multiplied by the normalization factor for the tissue tested. This normalization method was used for all tissues except those derived from the Human Blood Fractions MTC Panel, which showed dramatic variation in some housekeeping genes depending on whether the tissue had been activated or not. In these tissues, normalization was carried out with a single housekeeping gene, beta-2-microglobulin. Results are given in FIGS. 8 and 9, showing the experimentally obtained copy numbers of mRNA per 10 ng of first-strand cDNA on the left and the normalized values on the right. RNAs used for the cDNA synthesis, along with their supplier and catalog numbers are shown in tables 3 and 4. TABLE 3 Whole-body-screen tissues Tissue Supplier Panel name and catalog number  1. brain Clontech Human Total RNA Panel I, K4000-1  2. heart Clontech Human Total RNA Panel I, K4000-1  3. kidney Clontech Human Total R.NA Panel I, K4000-1  4. liver Clontech Human Total RNA Panel I, K4000-1  5. lung Clontech Human Total KNA Panel I, K4000-1  6. trachea Clontech Human Total RNA Panel I, K4000-1  7. bone marrow Clontech Human Total KNA Panel II, K4001-1  8. colon Clontech Human Total RNA Panel II, K4001-1  9. small intestine Clontech Human Total RNA Panel II, K4001-1 10. spleen Clontech Human Total RNA Panel II, K4001-1 11. stomach Clontech Human Total RNA Panel II, K4001-1 12. thymus Clontech Human Total RNA Panel II, K4001-1 13. mammary gland Clontech Human Total RNA Panel III, K4002-1 14. skeletal muscle Clontech Human Total RNA Panel III, K4002-1 15. prostate Clontech Human Total RNA Panel III, K4002-1 16. testis Clontech Human Total RNA Panel III, K4002-1 17. uterus Clontech Human Total RNA Panel III, K4002-1 18. cerebellum Clontech Human Total RNA Panel IV, K4003-1 19. fetal brain Clontech Human Total RNA Panel IV, K4003-1 20. fetal liver Clontech Human Total RNA Panel IV, K4003-1 21. spinal cord Clontech Human Total RNA Panel IV, K4003-1 22. placenta Clontech Human Total RNA Panel IV, K4003-1 23. adrenal gland Clontech Human Total RNA Panel V, K4004-1 24. pancreas Clontech Human Total RNA Panel V, K4004-1 25. salivary gland Clontech Human Total RNA Panel V, K4004-1 26. thyroid Clontech Human Total RNA Panel V, K4004-1

[0360] TABLE 4 Blood/lung-screen tissues Tissue Supplier Panel name and catalog number  1. lymph node Clontech Human Immune System MTC Panel, K1426-1  2. peripheral blood Clontech Human immune System MTC Panel, leukocytes K1426-1  3. tonsil Clontech Human Immune System MTC Panel, K1426-1  4. peripheral blood Clontech Human Blood Fractions MTC Panel, mononuclear cells K1428-1  5. peripheral blood Clontech Human Blood Fractions MTC Panel, mononuclear cells K1428-1 activated  6. T-cell (CD8+) Clontech Human Blood Fractions MTC Panel, K1428-1  7. T-cell (CD8+) Clontech Human Blood Fractions MTC Panel, activated K1428-1  8. T-cell (CD4+) Clontech Human Blood Fractions MTC Panel, K1428-1  9. T-cell (CD4+) Clontech Human Blood Fractions MTC Panel, activated K1428-1 10. B-cell (CD 19+) Clontech Human Blood Fractions MTC Panel, K1428-1 11. B-cell (CD19+) Clontech Human Blood Fractions MTC Panel, activated K1428-1 12. Monocytes Clontech Human Blood Fractions MTC Panel, (CD14+) K1428-1 13. Th1 clone In-house 14. Th2 clone In-house 15. neutrophil In-house 16. neutrophil In-house 17. Normal In-house Bronchial/Tracheal Epithelial Cells 18. Normal In-house Bronchial/Tracheal smooth muscle cell 19. Normal lung In-house fibroblast 20. Microvascular In-house Endothelial cell 21. U937 In-house 22. RAMOS In-house 23. Jurkat In-house 24. HelaS3 In-house 25. IMR-90 In-house 26. HEK293 In-house

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[0366] Liew, C. C., Hwang, D. M., Fung, Y. W., Laurenson, C., Cukerman, E., Tsui, S. & Lee, C. Y. (1994) A catalog of genes in the cardiovascular system as identified by expressed sequence tags. Proc. Natl. Acad. Sci. USA 91:10145-10649.

1 4 1 568 DNA Homo sapiens misc_feature (90)..(90) n=a, c, g or t 1 gtgcccctcc accacctctc catccaagga agccacgaca cgatgacgta ctgcctgaac 60 aagaagtccg ccatgtcgca cgaggagtcn cggctgctgc agctgctgaa caaggccttg 120 ccctgcatca cgcgccctgt cgtgctgaaa tggtccgtca cccaggcact ggacgtcaca 180 gagcagctgg atgccggggt gcggtacctg gatctgcgga tagcccacat gctggagggc 240 tcggagaaga acctgcactt tgtccatatg gtgtacacaa cggcgctggt ggaggacaca 300 ctcacggaaa tctcggagtg gctggagcgg catccacgcg aggtggtcat cctggcctgc 360 agaaacttcg aggggctgag cgaggacctg cacgagtacc tggtcgcctg tatcaagaac 420 atcttcgggg acatgctgtg tcctcgtggg gaggtgccga cactgcggca gctgtggtcc 480 cggggccaac aggtcatcgt ctcctatgaa gacgagagct ccttgcgccg gcaccacgag 540 ctgtggccag gagtccccta ctggtggg 568 2 323 PRT Homo sapiens 2 Met Gly Gly Gln Val Ser Ala Ser Asn Ser Phe Ser Arg Leu His Cys 1 5 10 15 Arg Asn Ala Asn Glu Asp Trp Met Ser Ala Leu Cys Pro Arg Leu Trp 20 25 30 Asp Val Pro Leu His His Leu Ser Ile Pro Gly Ser His Asp Thr Met 35 40 45 Thr Tyr Cys Leu Asn Lys Lys Ser Pro Ile Ser His Glu Glu Ser Arg 50 55 60 Leu Leu Gln Leu Leu Asn Lys Ala Leu Pro Cys Ile Thr Arg Pro Val 65 70 75 80 Val Leu Lys Trp Ser Val Thr Gln Ala Leu Asp Val Thr Glu Gln Leu 85 90 95 Asp Ala Gly Val Arg Tyr Leu Asp Leu Arg Ile Ala His Met Leu Glu 100 105 110 Gly Ser Glu Lys Asn Leu His Phe Val His Met Val Tyr Thr Thr Ala 115 120 125 Leu Val Glu Asp Thr Leu Thr Glu Ile Ser Glu Trp Leu Glu Arg His 130 135 140 Pro Arg Glu Val Val Ile Leu Ala Cys Arg Asn Phe Glu Gly Leu Ser 145 150 155 160 Glu Asp Leu His Glu Tyr Leu Val Ala Cys Ile Lys Asn Ile Phe Gly 165 170 175 Asp Met Leu Cys Pro Arg Gly Glu Val Pro Thr Leu Arg Gln Leu Trp 180 185 190 Ser Arg Gly Gln Gln Val Ile Val Ser Tyr Glu Asp Glu Ser Ser Leu 195 200 205 Arg Arg His His Glu Leu Trp Pro Gly Val Pro Tyr Trp Trp Gly Asn 210 215 220 Arg Val Lys Thr Glu Ala Leu Ile Arg Tyr Leu Glu Thr Met Lys Ser 225 230 235 240 Cys Gly Arg Pro Gly Gly Leu Phe Val Ala Gly Ile Asn Leu Thr Glu 245 250 255 Asn Leu Gln Tyr Val Leu Ala His Pro Ser Glu Ser Leu Glu Lys Met 260 265 270 Thr Leu Pro Asn Leu Pro Arg Leu Ser Ala Trp Val Arg Glu Gln Cys 275 280 285 Pro Gly Pro Gly Ser Arg Cys Thr Asn Ile Ile Ala Gly Asp Phe Ile 290 295 300 Gly Ala Asp Gly Phe Val Ser Asp Val Ile Ala Leu Asn Gln Lys Leu 305 310 315 320 Leu Trp Cys 3 317 PRT Listeria monocytogenes 3 Met Tyr Lys Asn Tyr Leu Gln Arg Thr Leu Val Leu Leu Leu Cys Phe 1 5 10 15 Ile Leu Tyr Phe Phe Thr Phe Pro Leu Gly Gly Lys Ala Tyr Ser Leu 20 25 30 Asn Asn Trp Asn Lys Pro Ile Lys Asn Ser Val Thr Thr Lys Gln Trp 35 40 45 Met Ser Ala Leu Pro Asp Thr Thr Asn Leu Ala Ala Leu Ser Ile Pro 50 55 60 Gly Thr His Asp Thr Met Ser Tyr Asn Gly Asp Ile Thr Trp Thr Leu 65 70 75 80 Thr Lys Pro Leu Ala Gln Thr Gln Thr Met Ser Leu Tyr Gln Gln Leu 85 90 95 Glu Ala Gly Ile Arg Tyr Ile Asp Ile Arg Ala Lys Asp Asn Leu Asn 100 105 110 Ile Tyr His Gly Pro Ile Phe Leu Asn Ala Ser Leu Ser Gly Val Leu 115 120 125 Glu Thr Ile Thr Gln Phe Leu Lys Lys Asn Pro Lys Glu Thr Ile Ile 130 135 140 Met Arg Leu Lys Asp Glu Gln Asn Ser Asn Asp Ser Phe Asp Tyr Arg 145 150 155 160 Ile Gln Pro Leu Ile Asn Ile Tyr Lys Asp Tyr Phe Tyr Thr Thr Pro 165 170 175 Arg Thr Asp Thr Ser Asn Lys Ile Pro Thr Leu Lys Asp Val Arg Gly 180 185 190 Lys Ile Leu Leu Leu Ser Glu Asn His Thr Lys Lys Pro Leu Val Ile 195 200 205 Asn Ser Arg Lys Phe Gly Met Gln Phe Gly Ala Pro Asn Gln Val Ile 210 215 220 Gln Asp Asp Tyr Asn Gly Pro Ser Val Lys Thr Lys Phe Lys Glu Ile 225 230 235 240 Val Gln Thr Ala Tyr Gln Ala Ser Lys Ala Asp Asn Lys Leu Phe Leu 245 250 255 Asn His Ile Ser Ala Thr Ser Leu Thr Phe Thr Pro Arg Gln Tyr Ala 260 265 270 Ala Ala Leu Asn Asn Lys Val Glu Gln Phe Val Leu Asn Leu Thr Ser 275 280 285 Glu Lys Val Arg Gly Leu Gly Ile Leu Ile Met Asp Phe Pro Glu Lys 290 295 300 Gln Thr Ile Lys Asn Ile Ile Lys Asn Asn Lys Phe Asn 305 310 315 4 972 DNA Homo sapiens 4 atgggtgggc aggtgagcgc ttccaacagc ttctcgaggc tgcactgcag aaatgccaac 60 gaggactgga tgtcggcact gtgtccccgg ctctgggatg tgcccctcca ccacctctcc 120 atcccaggga gccacgacac gatgacgtac tgcctgaaca agaagtcccc catttctcac 180 gaggagtccc ggctgctgca gctgctgaac aaggccttgc cctgcatcac gcgccctgtc 240 gtgctgaaat ggtccgtcac ccaggcactg gacgtcacag agcagctgga tgccggggtg 300 cggtacctgg acctgcggat agcccacatg ctggagggct cggagaagaa cctgcacttt 360 gtccatatgg tgtacacaac ggcgctggtg gaggacacac tcacggaaat ctcggagtgg 420 ctggagcggc atccacgcga ggtggtcatc ctggcctgca gaaacttcga ggggctgagc 480 gaggacctgc acgagtacct ggtcgcctgt atcaagaaca tcttcgggga catgctgtgt 540 cctcgtgggg aggtgccgac actgcggcag ctgtggtccc ggggccaaca ggtcatcgtc 600 tcctatgaag acgagagctc cttgcgccgg caccacgagc tgtggccagg agtcccctac 660 tggtggggaa acagggtgaa gaccgaggcc ctcatccgat acctggagac catgaagagc 720 tgcggccgcc caggagggtt gttcgtggcc ggcatcaacc tcacggagaa cctgcagtac 780 gttctggcgc acccgtccga gtccctggag aagatgacgc tgcccaacct tccgcggctg 840 agcgcgtggg tccgagagca gtgcccgggg ccgggttcac ggtgcaccaa catcatcgcg 900 ggggacttca tcggcgcaga cggcttcgtc agtgacgtca tcgcgctcaa tcagaagctg 960 ctgtggtgct ga 972 

1. An isolated polynucleotide encoding a PI-PLC-like enzyme polypeptide and being selected from the group consisting of: a) a polynucleotide encoding a PI-PLC-like enzyme polypeptide comprising an amino acid sequence selected form the group consisting of: amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 2; and the amino acid sequence shown in SEQ ID NO:
 2. b) a polynucleotide comprising the sequence of SEQ ID NOs: 1 or 4; c) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b); d) a polynucleotide the sequence of which deviates from the polynucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code; and e) a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a to (d).
 2. An expression vector containing any polynucleotide of claim
 1. 3. A host cell containing the expression vector of claim
 2. 4. A substantially purified PI-PLC-like enzyme polypeptide encoded by a polynucleotide of claim
 1. 5. A method for producing a PI-PLC-like enzyme polypeptide, wherein the method comprises the following steps: a) culturing the host cell of claim 3 under conditions suitable for the expression of the PI-PLC-like enzyme polypeptide; and b) recovering the PI-PLC-like enzyme polypeptide from the host cell culture.
 6. A method for detection of a polynucleotide encoding a PI-PLC-like enzyme polypeptide in a biological sample comprising the following steps: a) hybridizing any polynucleotide of claim 1 to a nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting said hybridization complex.
 7. The method of claim 6, wherein before hybridization, the nucleic acid material of the biological sample is amplified.
 8. A method for the detection of a polynucleotide of claim 1 or a PI-PLC-like enzyme polypeptide of claim 4 comprising the steps of: contacting a biological sample with a reagent which specifically interacts with the polynucleotide or the PI-PLC-like enzyme polypeptide.
 9. A diagnostic kit for conducting the method of any one of claims 6 to
 8. 10. A method of screening for agents which decrease the activity of a PI-PLC-like enzyme, comprising the steps of: contacting a test compound with any PI-PLC-like enzyme polypeptide encoded by any polynucleotide of claim 1; detecting binding of the test compound to the PI-PLC-like enzyme polypeptide, wherein a test compound which binds to the polypeptide is identified as a potential therapeutic agent for decreasing the activity of a PI-PLC-like enzyme.
 11. A method of screening for agents which regulate the activity of a PI-PLC-like enzyme, comprising the steps of: contacting a test compound with a PI-PLC-like enzyme polypeptide encoded by any polynucleotide of claim 1; and detecting a PI-PLC-like enzyme activity of the polypeptide, wherein a test compound which increases the PI-PLC-like enzyme activity is identified as a potential therapeutic agent for increasing the activity of the PI-PLC-like enzyme, and wherein a test compound which decreases the PI-PLC-like enzyme activity of the polypeptide is identified as a potential therapeutic agent for decreasing the activity of the PI-PLC-like enzyme.
 12. A method of screening for agents which decrease the activity of a PI-PLC-like enzyme, comprising the steps of: contacting a test compound with any polynucleotide of claim 1 and detecting binding of the test compound to the polynucleotide, wherein a test compound which binds to the polynucleotide is identified as a potential therapeutic agent for decreasing the activity of PI-PLC-like enzyme.
 13. A method of reducing the activity of PI-PLC-like enzyme, comprising the steps of: contacting a cell with a reagent which specifically binds to any polynucleotide of claim 1 or any PI-PLC-like enzyme polypeptide of claim 4, whereby the activity of PI-PLC-like enzyme is reduced.
 14. A reagent that modulates the activity of a PI-PLC-like enzyme polypeptide or a polynucleotide wherein said reagent is identified by the method of any of the claim 10 to
 12. 15. A pharmaceutical composition, comprising: the expression vector of claim 2 or the reagent of claim 14 and a pharmaceutically acceptable carrier.
 16. Use of the pharmaceutical composition of claim 15 for modulating the activity of a PI-PLC-like enzyme in a disease.
 17. Use of claim 16 wherein the disease is cancer, peripheral or central nervous system disease, chronic obstructive pulmonary disease or asthma.
 18. A cDNA encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO:2.
 19. The cDNA of claim 18 which comprises SEQ ID NOS:1 or
 4. 20. The cDNA of claim 18 which consists of SEQ ID NOS:1 or
 4. 21. An expression vector comprising a polynucleotide which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO:2.
 22. The expression vector of claim 21 wherein the polynucleotide consists of SEQ ID NOS:1 or
 4. 23. A host cell comprising an expression vector which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO:2.
 24. The host cell of claim 23 wherein the polynucleotide consists of SEQ ID NOS:1 or
 4. 25. A purified polypeptide comprising the amino acid sequence shown in SEQ ID NO:2.
 26. The purified polypeptide of claim 25 which consists of the amino acid sequence shown in SEQ ID NO:2.
 27. A fusion protein comprising a polypeptide having the amino acid sequence shown in SEQ ID NO:2.
 28. A method of producing a polypeptide comprising the amino acid sequence shown in SEQ ID NO:2, comprising the steps of: culturing a host cell comprising an expression vector which encodes the polypeptide under conditions whereby the polypeptide is expressed; and isolating the polypeptide.
 29. The method of claim 28 wherein the expression vector comprises SEQ ID NOS:1 or
 4. 30. A method of detecting a coding sequence for a polypeptide comprising the amino acid sequence shown in SEQ ID NO:2, comprising the steps of: hybridizing a polynucleotide comprising 11 contiguous nucleotides of SEQ ID NOS:1 or 4 to nucleic acid material of a biological sample, thereby forming a hybridization complex; and detecting the hybridization complex.
 31. The method of claim 30 further comprising the step of amplifying the nucleic acid material before the step of hybridizing.
 32. A kit for detecting a coding sequence for a polypeptide comprising the amino acid sequence shown in SEQ ID NO:2, comprising: a polynucleotide comprising 11 contiguous nucleotides of SEQ ID NOS:1 or 4; and instructions for the method of claim
 30. 33. A method of detecting a polypeptide comprising the amino acid sequence shown in SEQ ID NO:2, comprising the steps of: contacting a biological sample with a reagent that specifically binds to the polypeptide to form a reagent-polypeptide complex; and detecting the reagent-polypeptide complex.
 34. The method of claim 33 wherein the reagent is an antibody.
 35. A kit for detecting a polypeptide comprising the amino acid sequence shown in SEQ ID NO:2, comprising: an antibody which specifically binds to the polypeptide; and instructions for the method of claim
 33. 36. A method of screening for agents which can modulate the activity of a human PI-PLC-like enzyme, comprising the steps of: contacting a test compound with a polypeptide comprising an amino acid sequence selected from the group consisting of: (1) amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO:2 and (2) the amino acid sequence shown in SEQ ID NO:2; and detecting binding of the test compound to the polypeptide, wherein a test compound which binds to the polypeptide is identified as a potential agent for regulating activity of the human PI-PLC-like enzyme.
 37. The method of claim 36 wherein the step of contacting is in a cell.
 38. The method of claim 36 wherein the cell is in vitro.
 39. The method of claim 36 wherein the step of contacting is in a cell-free system.
 40. The method of claim 36 wherein the polypeptide comprises a detectable label.
 41. The method of claim 36 wherein the test compound comprises a detectable label.
 42. The method of claim 36 wherein the test compound displaces a labeled ligand which is bound to the polypeptide.
 43. The method of claim 36 wherein the polypeptide is bound to a solid support.
 44. The method of claim 36 wherein the test compound is bound to a solid support.
 45. A method of screening for agents which modulate an activity of a human PI-PLC-like enzyme, comprising the steps of: contacting a test compound with a polypeptide comprising an amino acid sequence selected from the group consisting of: (1) amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO:2 and (2) the amino acid sequence shown in SEQ ID NO:2; and detecting an activity of the polypeptide, wherein a test compound which increases the activity of the polypeptide is identified as a potential agent for increasing the activity of the human PI-PLC-like enzyme, and wherein a test compound which decreases the activity of the polypeptide is identified as a potential agent for decreasing the activity of the human PI-PLC-like enzyme.
 46. The method of claim 45 wherein the step of contacting is in a cell.
 47. The method of claim 45 wherein the cell is in vitro.
 48. The method of claim 45 wherein the step of contacting is in a cell-free system.
 49. A method of screening for agents which modulate an activity of a human PI-PLC-like enzyme, comprising the steps of: contacting a test compound with a product encoded by a polynucleotide which comprises the nucleotide sequence shown in SEQ ID NOS:1 or 4; and detecting binding of the test compound to the product, wherein a test compound which binds to the product is identified as a potential agent for regulating the activity of the human PI-PLC-like enzyme.
 50. The method of claim 49 wherein the product is a polypeptide.
 51. The method of claim 49 wherein the product is RNA.
 52. A method of reducing activity of a human PI-PLC-like enzyme, comprising the step of: contacting a cell with a reagent which specifically binds to a product encoded by a polynucleotide comprising the nucleotide sequence shown in SEQ ID NOS:1 or 4, whereby the activity of a human PI-PLC-like enzyme is reduced.
 53. The method of claim 52 wherein the product is a polypeptide.
 54. The method of claim 53 wherein the reagent is an antibody.
 55. The method of claim 52 wherein the product is RNA.
 56. The method of claim 55 wherein the reagent is an antisense oligonucleotide.
 57. The method of claim 56 wherein the reagent is a ribozyme.
 58. The method of claim 52 wherein the cell is in vitro.
 59. The method of claim 52 wherein the cell is in vivo.
 60. A pharmaceutical composition, comprising: a reagent which specifically binds to a polypeptide comprising the amino acid sequence shown in SEQ ID NO:2; and a pharmaceutically acceptable carrier.
 61. The pharmaceutical composition of claim 60 wherein the reagent is an antibody.
 62. A pharmaceutical composition, comprising: a reagent which specifically binds to a product of a polynucleotide comprising the nucleotide sequence shown in SEQ ID NOS:1 or 4; and a pharmaceutically acceptable carrier.
 63. The pharmaceutical composition of claim 62 wherein the reagent is a ribozyme.
 64. The pharmaceutical composition of claim 62 wherein the reagent is an antisense oligonucleotide.
 65. The pharmaceutical composition of claim 62 wherein the reagent is an antibody.
 66. A pharmaceutical composition, comprising: an expression vector encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO:2; and a pharmaceutically acceptable carrier.
 67. The pharmaceutical composition of claim 66 wherein the expression vector comprises SEQ ID NOS:1 or
 4. 68. A method of treating a PI-PLC-like enzyme disfunction related disease, wherein the disease is selected from cancer, peripheral or central nervous system disease, chronic obstructive pulmonary disease or asthma, comprising the step of: administering to a patient in need thereof a therapeutically effective dose of a reagent that modulates a function of a human PI-PLC-like enzyme, whereby symptoms of the PI-PLC-like enzyme disfunction related disease are ameliorated.
 69. The method of claim 68 wherein the reagent is identified by the method of claim
 36. 70. The method of claim 68 wherein the reagent is identified by the method of claim
 45. 71. The method of claim 68 wherein the reagent is identified by the method of claim
 49. 